Therapeutic agents and uses thereof

ABSTRACT

The present application provides an agent comprising or consisting of a binding moiety with specificity for a kallikrein protein (for example, PSA or hK2) for use in the treatment of prostate cancer, and a method for the treatment of prostate cancer in a patient, the method comprising the step of administering a therapeutically effective amount of an agent comprising or consisting of a binding moiety with specificity for a kallikrein protein to the patient.

PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/135,028, filed Apr. 21, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/354,617, filed Apr. 28, 2014, which is a § 371application of PCT/GB2012/052675, filed Oct. 26, 2012, which in turnclaims priority to U.S. Provisional Application 61/552,796, filed Oct.28, 2011. The entire disclosure of each of the foregoing applications isincorporated by reference herein.

FIELD OF THE INVENTION

This invention pertains in general to the field of therapeutic agentsand methods, particularly in field of prostate cancer.

BACKGROUND

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

Prostate cancer is at the present time the most common form of canceramong men. The prostate is a walnut-sized gland in men that producesfluid that is a component in semen. The prostate has two, or more,lobes, or sections, enclosed by an outer layer of tissue. The prostateis located in front of the rectum and just below the urine bladder, andsurrounds the urethra.

The occurrence of prostate cancer is highest in the northwestern part ofEurope and in the United States. The growth of the tumor is usually aprocess that takes place during a long period of time. Prostate canceris normally a mild form of cancer. In fact, the majority of mendiagnosed with prostate cancer do survive, and only a minority of themen encounters a more aggressive form of prostate cancer, whichmetastasizes in an early stage. This form of prostate cancer may only becurable if it is diagnosed in an early stage, before the cancer hasspread to extracapsular tissue.

Today diagnosis and monitoring of prostate cancer may be performed bymeasuring the concentration of a prostate specific antigen (PSA) in theblood of the patient. If the concentration of PSA is markedly high inseveral consecutive measurements, performed at different points of time,the assessment is that there is a probability of prostate cancer. Atthis point of time a biopsy may be performed to verify prostate cancer.

PSA (also known as kallikrein III) is a protein, constituted of a singlechain of 237 amino acids, that is produced in the secretory cells of theprostate. These secretory cells may be found in the whole prostategland. PSA is well established and thoroughly researched marker inrespect of prostate cancer. By comparison with healthy cells theproduction of PSA is lower in malignant cells and higher in hyperplasticcells. It is therefore contradicting that in fact the concentration ofPSA is higher in blood from men suffering from prostate cancer. However,one explanation may be that the malignant cells have a deteriorated cellstructure, and are therefore more permeable to PSA.

Another important serine protease, which may be suitable for futuretherapy of prostate cancer, is human glandular kallikrein 2 (hK2). Thegene coding hK2 is located on chromosome 19, together with the genecoding for PSA. hK2 is expressed mainly in the prostate tissue, just asPSA. In the prostate, PSA is present as an inactive pro-form and isactivated through the peptidase action of hK2. Immunohistochemicalresearch in respect of hK2 has shown that hK2 is expressed in relationto the level of differentiation. This means that hK2 is expressed in ahigher yield in tissue of low differentiation, such as tissue subjectedto prostate cancer, and in a lower yield in tissue of highdifferentiation, such as tissue subjected to benign prostatichyperplasia (BPH) which is another common prostate problem.

Today's therapies of prostate cancer are surgery (e.g., radicalprostatectomy), radiation therapy (including, brachytherapy and externalbeam radiation therapy, high-intensity focused ultrasound (HIFU),chemotherapy, oral chemotherapeutic drugs, cryosurgery (freezing thetumor), hormonal therapy (such as antiandrogen therapy), castration orcombinations of the foregoing.

Most of these therapies (surgery and external radiation therapy) are,however, only (or primarily) useful for treatment of primary tumors andlarge metastases. Chemotherapy is used for disseminated of the cancerbut for most of these patients, it is a palliative effect and/orprolonged survival. Other or complementary treatment modalities aretherefore necessary to achieve considerable improvements of thedisseminated malignant diseases, particular in cases of micrometastases.

Therapy, such as immunotherapy or radioimmunotherapy, using targetingmolecules such as antibodies and fragments could give the possibility oftherapy of disseminated disease.

Thus, there is a need for a new therapeutic agents and methods fortreating prostate cancer, particular in cases of disseminated disease,metastases and micrometastases.

SUMMARY OF THE INVENTION

Accordingly, the present invention seeks to mitigate, alleviate oreliminate one or more of the above-identified deficiencies in the artand disadvantages singly or in any combination and solves at least theabove-mentioned problems by providing a therapy method according to theappended patent claims.

A first aspect, present invention provides an agent comprising orconsisting of a binding moiety with specificity for a kallikrein proteinfor use in the treatment of prostate cancer.

To put it another way, the first aspect of the present invention relatesto the use of an agent comprising or consisting of a binding moiety withspecificity for a kallikrein protein in the manufacture of a medicamentfor the treatment of prostate cancer.

Accordingly, first aspect also provides a method for the treatment ofprostate cancer in a patient, the method comprising the step ofadministering a therapeutically effective amount of an agent comprisingor consisting of a binding moiety with specificity for a kallikreinprotein.

By “binding moiety” we include all types of chemical entity (forexample, oligonucleotides, polynucleotide, polypeptides, peptidomimeticsand small compounds) which are capable of binding specifically to akallikrein protein. Advantageously, the binding moiety is capable ofbinding selectively (i.e., preferentially) to a kallikrein protein underphysiological conditions.

As indicated above, the agents of the invention may comprise or consistof any suitable chemical entity constituting a binding moiety withspecificity for a kallikrein protein.

Methods suitable for detecting interactions between a test chemicalentity and a kallikrein protein are well known in the art. For example,ultrafiltration with ion spray mass spectroscopy/HPLC methods or otherphysical and analytical methods may be used. In addition, FluorescenceEnergy Resonance Transfer (FRET) methods may be used, in which bindingof two fluorescent labelled entities may be measured by measuring theinteraction of the fluorescent labels when in close proximity to eachother.

Alternative methods of detecting binding of a kallikrein protein tomacromolecules, for example DNA, RNA, proteins and phospholipids,include a surface plasmon resonance assay, for example as described inPlant et al., 1995, Analyt Biochem 226(2), 342-348. Such methods maymake use of a polypeptide that is labelled, for example with aradioactive or fluorescent label.

A further method of identifying a chemical entity that is capable ofbinding to a kallikrein protein is one where the kallikrein protein isexposed to the compound and any binding of the compound to the saidkallikrein protein is detected and/or measured. The binding constant forthe binding of the compound to the kallikrein protein may be determined.Suitable methods for detecting and/or measuring (quantifying) thebinding of a compound to a kallikrein protein are well known to thoseskilled in the art and may be performed, for example, using a methodcapable of high throughput operation, for example a chip-based method.New technology, called VLSIPS™, has enabled the production of extremelysmall chips that contain hundreds of thousands or more of differentmolecular probes. These biological chips have probes arranged in arrays,each probe assigned a specific location. Biological chips have beenproduced in which each location has a scale of, for example, tenmicrons. The chips can be used to determine whether target moleculesinteract with any of the probes on the chip. After exposing the array totarget molecules under selected test conditions, scanning devices canexamine each location in the array and determine whether a targetmolecule has interacted with the probe at that location.

Another method of identifying compounds with binding affinity for akallikrein protein is the yeast two-hybrid system, where thepolypeptides of the invention can be used to “capture” proteins thatbind the kallikrein protein. The yeast two-hybrid system is described inFields & Song, Nature 340:245-246 (1989).

In one embodiment, the binding moiety may comprise or consist of apolypeptide.

For example, the binding moiety may comprise or consist of an antibodyor an antigen-binding fragment thereof with binding specificity for akallikrein protein, or a variant, fusion or derivative of said antibodyor antigen-binding fragment, or a fusion of a said variant or derivativethereof, which retains the binding specificity for the kallikreinprotein.

Thus, in one embodiment of the first aspect of the present invention,the binding moiety may be an antibody or antigen-binding fragmentthereof.

By “antibody” we include substantially intact antibody molecules, aswell as chimaeric antibodies, humanised antibodies, human antibodies(wherein at least one amino acid is mutated relative to the naturallyoccurring human antibodies), single chain antibodies, diabodies,bispecific antibodies, antibody heavy chains, antibody light chains,homodimers and heterodimers of antibody heavy and/or light chains, andantigen binding fragments and derivatives of the same.

By “antigen-binding fragment” we mean a functional fragment of anantibody that is capable of binding to a kallikrein protein. The bindingaffinity of the different antibody derivatives mentioned above may bedetermined with Scatchard's method using a fixed concentration ofimmobilized antibody fragment and varying concentrations of Eu-PSAtracer. Alternatively, the binding affinity may be determined usingSurface Plasmon resonance (SPR) technology on a Biacore instrument. Theanalysis methods are further described in Example 8.

In particular, the antigen-binding fragment is selected from the groupconsisting of Fv fragments (e.g., single chain Fv and disulphide-bondedFv), Fab-like fragments (e.g., Fab fragments, Fab′ fragments and F(ab)₂fragments), single variable domains (e.g., V_(H) and V_(L) domains) anddomain antibodies (dAbs, including single and dual formats [i.e.,dAb-linker-dAb]).

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue and/or faster blood clearance which may permit highertherapeutic ratios. Moreover, antigen-binding fragments such as Fab, Fv,ScFv and dAb antibody fragments can be expressed in and secreted frommicroorganisms, such as E. coli, thus allowing the facile production oflarge amounts of the said fragments.

Also included within the scope of the invention are modified versions ofantibodies and antigen-binding fragments thereof, e.g., modified by thecovalent attachment of polyethylene glycol or other suitable polymer(see below).

Methods of generating antibodies and antibody fragments are well knownin the art. For example, antibodies may be generated via any one ofseveral methods which employ induction of in vivo production of antibodymolecules, screening of immunoglobulin libraries (Orlandi. et al, 1989.Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991, Nature349:293-299) or generation of monoclonal antibody molecules by celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the Epstein-Barrvirus (EBV)-hybridoma technique (Kohler et al., 1975. Nature256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote etal., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984.Mol. Cell. Biol. 62:109-120).

Suitable monoclonal antibodies to selected antigens may be prepared byknown techniques, for example those disclosed in “Monoclonal Antibodies:A manual of techniques”, H Zola (CRC Press, 1988) and in “MonoclonalHybridoma Antibodies: Techniques and Applications”, J G R Hurrell (CRCPress, 1982).

Likewise, antibody fragments can be obtained using methods well known inthe art (see, for example, Harlow & Lane, 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory, New York). Forexample, antibody fragments according to the present invention can beprepared by proteolytic hydrolysis of the antibody or by expression inE. coli or mammalian cells (e.g., Chinese hamster ovary cell culture orother protein expression systems) of DNA encoding the fragment.Alternatively, antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods.

It will be appreciated by persons skilled in the art that for humantherapy or diagnostics, human or humanised antibodies may be used.Humanised forms of non-human (e.g., murine) antibodies are geneticallyengineered chimaeric antibodies or antibody fragments havingminimal-portions derived from non-human antibodies. Humanised antibodiesinclude antibodies in which complementary determining regions of a humanantibody (recipient antibody) are replaced by residues from acomplementary determining region of a non human species (donor antibody)such as mouse, rat of rabbit having the desired functionality. In someinstances, Fv framework residues of the human antibody are replaced bycorresponding non-human residues. Humanised antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported complementarity determining region or framework sequences. Ingeneral, the humanised antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the complementarity determining regions correspondto those of a non human antibody and all, or substantially all, of theframework regions correspond to those of a relevant human consensussequence. Humanised antibodies optimally also include at least a portionof an antibody constant region, such as an Fc region, typically derivedfrom a human antibody (see, for example, Jones et al., 1986. Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992,Curr. Op. Struct. Biol. 2:593-596).

Methods for humanising non-human antibodies are well known in the art.Generally, the humanised antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues, often referred to as imported residues, aretypically taken from an imported variable domain. Humanisation can beessentially performed as described (see, for example, Jones et al.,1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988, Science 239:1534-15361; U.S. Pat. No. 4,816,567)by substituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanised antibodies are chimaeric antibodies, whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanised antibodies may be typically human antibodies inwhich some complementarity determining region residues and possibly someframework residues are substituted by residues from analogous sites inrodent antibodies.

Human antibodies can also be identified using various techniques knownin the art, including phage display libraries (see, for example,Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies andCancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol.147:86-95).

Once suitable antibodies are obtained, they may be tested for activity,for example by ELISA.

In an alternative embodiment of the first aspect of the invention, thebinding moiety comprises or consists of a non-immunoglobulin bindingmoiety, for example as described in Skerra, Curr Opin Biotechnol. 2007August; 18(4):295-304.

In a further alternative embodiment, the binding moiety comprises orconsists of an aptamer. For example, the agent may comprise or consistof a peptide aptamer or a nucleic acid aptamer (see Hoppe-Seyler & Butz,2000, J Mol Med. 78 (8): 426-30; Bunka D H & Stockley P G, 2006, Nat RevMicrobiol. 4 (8): 588-96 and Drabovich et al., 2006, Anal Chem. 78 (9):3171-8).

In a still further alternative embodiment, the binding moiety comprisesor consists of a small chemical entity. Such entities with kallikreinbinding properties may be identified by screening commercial librariesof small compounds (for example, as available from ChemBridgeCorporation, San Diego, USA).

Accordingly, the binding moiety present in the agent according to thefirst aspect of the present invention binds with specificity for akallikrein protein. In this context, the phrase “binds with specificity”means that the binding moiety binds selectively to the target kallikreinprotein in preference to other proteins, optionally including otherkallikrein proteins. The skilled person is well aware of numerousmethods for assessing the binding specificity of a binding molecule to atarget. For example, where the binding molecule is, or is based on, anantibody, its ability to bind specifically to for a kallikrein proteinmay be assessed by an immunoassay, such as an ELISA, radioimmunoassay,or the like.

In one embodiment, the binding moiety may be said to bind withspecificity for a kallikrein protein if it binds to the kallikreinprotein in an immunoassay and/or under physiological conditions (such asconditions found in the prostate or other sites for treatment asdiscussed herein) with a binding affinity of greater than 1×10⁵, 1×10⁶,1×10⁷, 2×10⁷, 1×10⁸, 2×10⁸, 1×10⁹, 2×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 1×10¹¹or more, such as within the range of from 1×10⁵ to 3×10¹⁰, or more.

The binding moiety as used in the first aspect of the present inventionmay have specificity for a human kallikrein protein. A human kallikreinprotein is a serine protease belonging to the human tissue kallikreingene family which was found to consist of least 15 members (Hsieh M L,Cancer Res 1997; 57; 2651-6). Kallikreins are heat-stable glycoproteinswith a single polypeptide chain, with a Mw varying between 27-40 kDa.

The binding moiety as used in the first aspect of the present inventionmay have specificity for a kallikrein protein selected from the groupconsisting of prostate-specific antigen (PSA; hk3, human kallikrein 3)and human glandular kallikrein (hK2).

In one embodiment, the binding moiety has specificity for PSA. The termPSA is intended to include every known form of PSA, such as free PSA,precursor forms of PSA, internally nicked forms of PSA, low molecularweight free PSA, standard weight free PSA, inactive mature PSA,truncated forms of PSA, glycosylation variants of PSA, BPSA, inactivepro-PSA, and every complex of PSA, such as PSA bound toα1-antichymotrypsin (ACT), α1-protease inhibitor (API), andα2-macroglobulin (AMG). An exemplary primary amino acid of PSA isprovided in FIG. 14 (see SEQ ID NO:16).

PSA, secreted from cancer cells, is in a more active state in comparisonwith PSA, secreted from BPH tissue. In the extracellular fluid PSA maybe subjected to proteolytic degradation, thus leading to loss ofactivity and formation of complexes. Thus, it is also within the scopeof the present invention to label compounds or entities, such as ACT,API, and AMG, bound or complexed to/with PSA.

In one preferred embodiment, the binding moiety has specificity for thefree (that is, non-complexed) isoform of PSA compared to the complexedisoform of PSA. Binding moieties with specificity for the free isoformof PSA may have binding specificity for an epitope that is exposed onthe free isoform of PSA, but is unexposed on the complexed isoform ofPSA, such as a conformational (that is, non-linear) epitope. An exampleof such a conformational epitope is formed from amino acid residues thatare part of the kallikrein-loop surrounding the catalytic cleft of PSA,and may include the active site triad of His41, Asn96, and Ser189). SeeLeinonen et al, Clinical Chemistry 48:12, 2208-2216 (2002) for furtherdiscussion and disclosure of numerous suitable epitopes on PSA, whichare incorporated herein by reference.

Where the binding moiety as used in the first aspect of the presentinvention has specificity for PSA, then the binding moiety may competefor binding to PSA (such as the free isoform of PSA), or a peptidecomprising the reactive epitope of PSA as bound by the binding moiety,with an antibody selected from the group consisting of PSA30, 4D4, 5C3,and 5A10, and an antigen-binding fragment thereof. Further discussion ofsuch antibodies may be found in Pettersson et al, Clin. Chem, 41:10,1480-1488 (1995); Nilsson et al, Brit. J. Cancer, 75:6, 789-797 (1997);Leinonen et al, Clinical Chemistry 48:12, 2208-2216 (2002); Väisänen etal, Anal. Chem., 78:7809-7815 (2006); Evans-Axelsson et al., CancerBiother. Radiopharm. 27:4, 243-51, EP 1 320 756 B1; and US 2004/101914,the contents of each of which are incorporated herein by reference.

The amino acid sequence of the constituent heavy and light chains of theexemplary anti-PSA antibody 5A10 is shown below (in which the CDRsequences are underlined).

5A10 Heavy chain [SEQ ID NO: 1]EVQLVESGPGILQPSQTLSLTCSFSGFSLSTTGMGVSWIRQPSGKGLEWLAHLYWDEDKRYNPSLKSRLTISEDSSRNQVFLKITSVGPADSATYYCAR KGYYGYFDYWGQGTALTVSS5A10 Light chain [SEQ ID NO: 2]DIVMTQSQKFMSTSVGDRVSVTCKASQNVNTDVAWYQQKPGQSPKALIFSTSYRSSGVPDRFTGSGSGTDFTLTITNVQSEDLAEYFCQQYSNYPLT FGAGTKVDLN

In this context, the term “competes” includes the meaning that thepresence of the agent comprising the binding moiety in a competitiveassay along with an reference antibody selected from PSA30, 4D4, 5C3,and 5A10 can reduce the level of detectable binding of the referenceantibody to PSA (such as free PSA) by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (for example, when theagent and the reference antibody are present in the test in equimolaramounts, and optionally wherein the test is performed underphysiological conditions. Such analysis can be done by animmunoradiometric assay (IRMA) as described in Example 9.

Where the binding moiety as used in the first aspect of the presentinvention has specificity for PSA, then the binding moiety may comprisesone or more complementarity determining regions (CDRs) of an antibodyselected from the group consisting of PSA30, 4D4, 5C3, and 5A10 (asshown by the underlined sequences in SEQ ID NOS:1 to 8 above).

As is well established in the art, complete antibodies comprise sixCDRs, three of which are present in the variable light (V_(L)) change,and the other three of which are present in the variable heavy (V_(H))chain,

It is not necessary for binding molecules to containing all six of theCDRs of any of these antibodies in order to retain the antigen bindingactivity, although in one embodiment the binding molecule may compriseall six CDRs from an antibody selected from the group consisting ofPSA30, 4D4, 5C3, and 5A10.

Alternatively, the binding moiety may comprise less than six of theCDRs, such as:

-   -   five CDRs (i.e., 3 CDRs from the V_(H) or V_(L) region, 2 CDRs        from the other variable region);    -   four CDRs (i.e., 3 CDRs from the V_(H) or V_(L) region, 1 CDR        from the other variable region; or 2 CDRs from each of the V_(H)        and V_(L) regions);    -   three CDRs (i.e., all three CDRs from one of the V_(H) or V_(L)        regions, and none from the other; or 2 CDRs from the V_(H) or        V_(L) region, 1 CDR from the other variable region);    -   two CDRs (i.e., two CDRs from one of the V_(H) or V_(L) regions,        and none from the other; or 1 CDRs from each of the V_(H) and        V_(L) regions); or    -   one CDR from either of the V_(H) or V_(L) regions,        from an antibody selected from the group consisting of PSA30,        4D4, 5C3, and 5A10.

It is well known in the art that three or fewer CDR regions (in somecases, even just a single CDR or a part thereof) are capable ofretaining the antigen-binding activity of the antibody from which theCDR(s) are derived:

-   -   Laune et al. (1997), JBC, 272:30937-44—demonstrates that a range        of hexapeptides derived from a CDR display antigen-binding        activity (see abstract) and notes that synthetic peptides of a        complete, single, CDR displays strong binding activity (see page        30942, right-hand column).    -   Monnet et al. (1999), JBC, 274:3789-96—shows that a range of        12-mer peptides and associated framework regions have        antigen-binding activity (see abstract) and comments that a        CDR3-like peptide alone is capable of binding antigen (see page        3785, left-hand column).    -   Qiu et al. (2007), Nature Biotechnology, 25:921-9—demonstrates        that a molecule consisting of two linked CDRs are capable of        binding antigen (see abstract and page 926, right-hand column).    -   Ladner et al. (2007), Nature Biotechnology, 25:875-7—is a review        article reporting Qiu et al. (above) and commenting that        molecules containing two CDRs are capable of retaining        antigen-binding activity (see page 875, right-hand column).    -   Heap et al. (2005), J. Gen. Virol., 86:1791-1800—reports that a        “micro-antibody” (a molecule containing a single CDR) is capable        of binding antigen (see abstract and page 1791, left-hand        column) and shows that a cyclic peptide from an anti-HIV        antibody has antigen-binding activity and function.    -   Nicaise et al. (2004) Protein Science, 13:1882-91—shows that a        single CDR can confer antigen-binding activity and affinity for        its lysozyme antigen

Vaughan and Sollazzo (2001), Combinatorial Chemistry & High ThroughputScreening, 4:417-430—is a review article describing minibodies thatcontain less than three CDR regions. For example, on page 418 (rightcolumn—3 Our Strategy for Design) a minibody including only the H1 andH2 CDR hypervariable regions interspersed within framework regions isdescribed. The minibody is described as being capable of binding to atarget.

-   -   Quiocho (1993), Nature, 362:293-4—is a further review type        article that provides a summary of minibody technology (i.e.,        miniaturised antibodies—in this case with less than three CDRs).    -   Pessi et al (1993), Nature, 362:367-9 and Bianchi et al        (1994), J. Mol. Biol., 236:649-59—are papers referenced in the        Vaughan and Sollazzo review and describe the H1 and H2 minibody        and its properties in more detail.    -   Gao et al (1994), J. Biol. Chem., 269:32389-93 which describes a        whole V_(L) chain (including all three CDRs) having high        affinity for its substrate, the vasoactive intestinal peptide,        as evidence that it is not necessary to have both the V_(H) and        V_(L) chains.

These documents were published before the priority date of the presentapplication and would therefore have been available to the skilledperson when implement the present invention. They provide clear evidencethat molecules having fewer than all six CDRs can be capable ofretaining the antigen binding properties of the antibodies for whichthey are derived.

In one preferred embodiment, where the binding moiety as used in thefirst aspect of the present invention has specificity for PSA, then thebinding moiety is an antibody, or antigen-binding fragment or derivativethereof, comprising the six CDRs of exemplary anti-PSA antibody 5A10.

In an alternative embodiment, where the binding moiety as used in thefirst aspect of the present invention has specificity for PSA, then thebinding moiety may comprise or consist of an antibody selected from thegroup consisting of PSA30, 4D4, 5C3, and 5A10, and antigen-bindingfragments thereof.

In another embodiment of the first aspect of the present invention, thebinding moiety has specificity for human glandular kallikrein (hK2).

The term hK2 is intended to include all isomeric forms of hK2, and anymolecule or protein in complex with hK2. An exemplary hK2 sequence isdescribed as Transcript: KLK2-201 (ENST00000325321), a product of geneENSG00000167751, as given in the ensemble database which can be found atthe following world-wide-web address at:

ensembl.org/Homo_sapiens/Transcript/Sequence_Protein? g = ENSG00000167751;r = 19:51376689-51383822; t = ENST00000325321and has the following sequence:

[SEQ ID NO: 3] MWDLVLSIAL SVGCTGAVPL IQSRIVGGWE CEKHSQPWQVAVYSHGWAHC GGVLVHPQWV LTAAHCLKKN SQVWLGRHNLFEPEDTGQRV PVSHSFPHPL YNMSLLKHQS LRPDEDSSHDLMLLRLSEPA KITDVVKVLG LPTQEPALGT TCYASGWGSIEPEEFLRPRS LQCVSLHLLS NDMCARAYSE KVTEFMLCAGLWTGGKDTCG GDSGGPLVCN GVLQGITSWG PEPCALPEKP AVYTKVVHYR KWIKDTIAAN P

Most of the hK2 found in seminal plasma is inactive and complexed withprotein C inhibitor (PCI). It is also possible that hK2 forms complexeswith other extracellular protease inhibitors. In vitro studies show thathK2 may bind to α2-antiplasmin (α2-AP), ACT, AMG, anti-thrombin III(ATIII), C1-inactivator and plasminogen activator inhibitor-1 (PAI-1).

Thus, it is also within the scope of the present invention to labelcompounds, molecules, proteins or any other entity, such as PCI,α2-antiplasmin (α2-AP), ACT, AMG, anti-thrombin III (ATIII),C1-inactivator and plasminogen activator inhibitor-1 (PAI-1), bound orcomplexed to/with hK2.

In one embodiment, the binding moiety may have specificity for the free(that is, non-complexed) isoform of hK2 compared to the complexedisoform of hK2. Binding moieties with specificity for the free isoformof hK2 may have binding specificity for an epitope that is exposed onthe free isoform of hK2, but is unexposed on the complexed isoform ofhK2, and this may be a linear or a conformational (that is, non-linear)epitope.

For example the binding moiety may have specificity for an epitope thatincludes one or more amino acid residues that are part of the catalyticcleft of hK2 that is exposed in free hK2 and unexposed in a complexedisoform, such as the form present in seminal fluid when hK2 is complexedto PCI. Epitope mapping of hK2 is described in Väisänen et al, ClinicalChemistry 50:9, 1607-1617 (2004), the disclosures of which areincorporated herein by reference.

Where the binding moiety as used in the first aspect of the presentinvention has specificity for hK2, then the binding moiety may completefor binding to hK2, or a peptide comprising the reactive epitope of h2Kas bound by the binding moiety, with an antibody selected from the groupconsisting of 1166, and 7G1. Further discussion of such antibodies maybe found in Väisänen et al, Clinical Chemistry, 50:9, 1607-1617 (2004);and Väisänen et al, Anal. Chem., 78:7809-7815 (2006), the contents ofeach of which are incorporated herein by reference.

The amino acid sequence of the constituent heavy and light chains of theexemplary anti-hK2 antibody 11B6 is shown below (in which the CDRsequences are underlined).

11B6 Heavy chain [SEQ ID NO: 4]DVQLQESGPGLVKPSQSLSLTCTVTGNSITSDYAWNWIRQFPGNRLEWMGYISYSGSTTYSPSLKSRFSITRDTSKNQFFLQLNSVTPEDTATYFCAT GYYYGSGFWGQGTLVTVSS11B6 Light chain [SEQ ID NO: 5]DIVLTQSPASLAVSLGQRATISCRASESVEYFGTSLMHWYRQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIQPVEEDDFSMYFCQQTRKVPY TFGGGTKLEIK

In this context, the term “competes” includes the meaning that thepresence of the agent comprising the binding moiety in a competitiveassay along with an reference antibody selected from 11B6, and 7G1 canreduce the level of detectable binding of the reference antibody to hK2by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99% or 100% (for example, when the agent and the reference antibody arepresent in the test in equimolar amounts, and optionally wherein thetest is performed under physiological conditions). Such analysis can bedone by an immunoradiometric assay (IRMA) as described in Example 9.

Where the binding moiety as used in the first aspect of the presentinvention has specificity for hK2, then the binding moiety may compriseone or more complementarity determining regions (CDRs) of an antibodyselected from the group consisting of 11B6, and 7G1 (as shown by theunderlined sequences in SEQ ID NOS:10 to 13 above).

It is not necessary for binding molecules to containing all six of theCDRs of any of these antibodies in order to retain the antigen bindingactivity, although in one embodiment the binding molecule may compriseall six CDRs from an antibody selected from the group consisting of11B6, and 7G1.

Alternatively, the binding moiety may comprise less than six of theCDRs, such as—

-   -   five CDRs (i.e., 3 CDRs from the V_(H) or V_(L) region, 2 CDRs        from the other variable region);    -   four CDRs (i.e., 3 CDRs from the V_(H) or V_(L) region, 1 CDR        from the other variable region; or 2 CDRs from each of the V_(H)        and V_(L) regions);    -   three CDRs (i.e., all three CDRs from one of the V_(H) or V_(L)        regions, and none from the other; or 2 CDRs from the V_(H) or        V_(L) region, 1 CDR from the other variable region);    -   two CDRs (i.e., two CDRs from one of the V_(H) or V_(L) regions,        and none from the other; or 1 CDRs from each of the V_(H) and        V_(L) regions); or    -   one CDR from either of the V_(H) or V_(L) regions, from an        antibody selected from the group consisting of 11B6, and 7G1.

In one preferred embodiment, where the binding moiety as used in thefirst aspect of the present invention has specificity for hK2, then thebinding moiety is an antibody, or antigen-binding fragment or derivativethereof, comprising the six CDRs of exemplary anti-hK2 antibody 11B6(see underlined sequences of SEQ ID NOs: 4 and 5).

In an alternative embodiment, where the binding moiety as used in thefirst aspect of the present invention has specificity for hK2, then thebinding moiety may comprises or consists of an antibody selected fromthe group consisting of 11B6, and 7G1, and antigen-binding fragmentsthereof.

Optionally, the agent used in the first aspect of the present inventionmay further comprise a therapeutic moiety. Accordingly, the agent maycomprise, or consist, of a binding moiety as described above and atherapeutic moiety. The binding moiety may be linked directly, orindirectly, to the therapeutic moiety.

In the case that the agent may comprises, or consists, of a bindingmoiety as described above and a therapeutic moiety then the agent maydisplays tumour uptake characteristics, for example as tested under theconditions used in the examples below, substantially equivalent to thetumour uptake characteristics of an agent consisting of the bindingmoiety alone. In this context, substantially equivalent includes themeaning of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99% or substantially 100%.

Any suitable therapeutic moiety may be used. A suitable therapeuticmoiety is one that is capable of reducing or inhibiting the growth, orin particular killing, a prostatic cancer cell. For example, thetherapeutic agent may be a cytotoxic moiety. A cytotoxic moiety maycomprise or consist of one or more radioisotopes. For example, the oneor more radioisotopes may each independently selected from the groupconsisting of beta-emitters, Auger-emitters, conversionelectron-emitters, alpha-emitters, and low photon energy-emitters. Itmay be desired that the one or more radioisotopes each independently hasor have an emission pattern of locally absorbed energy that creates ahigh absorbed dose in the vicinity of the agent. Exemplary radioisotopesmay include long-range beta-emitters, such as ⁹⁰Y, ³²P, ¹⁸⁶Re/¹⁸⁸Re;¹⁶⁶Ho, ⁷⁶As/⁷⁷As, ⁸⁹Sr, ¹⁵³Sm; medium range beta-emitters, such as ¹³¹I,¹⁷⁷Lu, ⁶⁷Cu, ¹⁶¹Tb, ¹⁰⁵Rh; low-energy beta-emitters, such as ⁴⁵Ca or³⁵S; conversion or Auger-emitters, such as ⁵¹Cr, ⁶⁷Ga, ⁹⁹Tc^(m), ¹¹¹In,^(114m)In, ¹²³I, ¹²⁵I, ²⁰¹Tl; and alpha-emitters, such as ²¹²Bi, ²¹³Bi,²²³Ac, ²²⁵Ac, ²¹²Pb, ²⁵⁵Fm, ²²³Ra, ¹⁴⁹Tb and ²²¹At. Further examples oftherapeutic radionuclides can be seen in FIG. 9. Other radionuclides areavailable and will be possible to use for therapy. In anotherembodiment, it may be desired that the therapeutic moiety or cytotoxicmoiety is not a moiety as disclosed as a “tracer” in WO 2006/087374 A1,in particular at page 11, lines 7-15 thereof.

In one preferred embodiment, the therapeutic moiety is ¹⁷⁷Lu. Forexample, the agent may be an ¹¹¹Lu-labelled form of anti-hK2 antibody11B6, or of an antigen-binding fragment or derivative thereof.

Alternatively, the therapeutic moiety comprises or consists of one ormore therapeutic (such as cytotoxic) drugs, for example, a cytostaticdrug; an anti-androgen drug; cortisone and derivatives thereof; aphosphonate; a testosterone-5-α-reductase inhibitor; a boron addend; acytokine; thapsigargin and its metabolites; a toxin (such as saporin orcalicheamicin); a chemotherapeutic agent (such as an antimetabolite); orany other therapeutic or cytotoxic drug useful in the treatment ofprostatic carcinoma.

Exemplary therapeutic/cytotoxic drugs may, for example, include:

-   -   Cytostatics, in particular those with dose-limiting        side-effects, including but not limited to cyclophosamide,        chlorambucil, ifosfamide, busulphane, lomustine, taxanes,        estramustine phosphate and other nitrogen mustards, antibiotics        (including doxorubicine, calicheamicines and esperamicine),        vinca alkaloids, azaridines, platinum-containing compounds,        endostatin, alkyl sulfonates, nitrosoureas, triazenes, folic        acid analoges, pyrimidine analoges, purine analogs, enzymes,        substituted urea, methyl-hydrazi ne derivatives, daunorubicin,        amphipathic amines,    -   Anti-androgens such as flutamide and bikalutamide and        metabolites thereof;    -   Cortisone and derivatives thereof;    -   Phosphonates such as diphophonate and buphosphonate;    -   Testosterone-5-α-reductaseinhibitors;    -   Boron addends;    -   Cytokines;    -   Thapsigargin and its metabolites;    -   Other agents used in the treatment of prostatic carcinoma.

Alternatively, the cytotoxic moiety comprises or consists of one or moremoieties suitable for use in activation therapy, such as photonactivation therapy, neutron activation therapy, neutron induced Augerelectron therapy, synchrotron irradiation therapy or low energy X-rayphoton activation therapy.

For example, with the tumor targeting agents according to the presentinvention there will be the potential of using synchrotron radiation (orlow energy X-rays) for the advancement of radiotherapy, primarilyfocusing on so called photo-activation radiotherapy (PAT), in which thelocal energy deposition from external X-ray irradiation is enhanced inthe cancer tissue by the interaction with a pre-administered, high-Ztumor-targeting agent, see FIG. 10.

The PAT treatment modality utilises monochromatic X-rays from asynchrotron source, such as provided by the ID17 biomedical beamline atthe European Synchrotron Radiation Facility (ESRF) in Grenoble, and asanticipated to be available at other facilities in the future such asthe new Swedish synchrotron facility, Max-IV.

As a further potential treatment modality, research on “induced Augerelectron tumour therapy” is the coming European Spallation Source (ESS)in Lund, and hopefully a medical experimental station. Reactor-producedthermal and semi-thermal neutrons have for long been used forBoron-Neutron-Capture-Therapy, BNCT, both for pre-clinical experimentsand for treatment of brain tumours with the induced alpha-particles andthe recoil nucleus (⁷L) that give a high locally absorbed energy. Asimilar approach is to use neutrons and suitable tumour-targetingmolecules labelled with stable nuclei with high cross-section forneutrons. Antibodies or peptides can for instance be labelled withstable Gadolinium (¹⁵⁷Gd) and act as the target molecule for theneutrons that are captured by the Gd-nucleus, so called GadoliniumNeutron Capture Therapy (GdNCT). By Monte Carlo techniques, the dosedistribution in the tumour and the surrounding tissues is calculated asit results from γ-photons, neutrons, nuclear recoils, as well ascharacteristic x-rays, internal conversion and Auger-electrons fromgadolinium or other potential elements.

As discussed above, the therapeutic moiety (such as a radioisotope,cytotoxic moiety or the like) may be linked directly, or indirectly, tothe binding moiety (such as an antibody or fragment thereof). Suitablelinkers are known in the art and include, for example, prostheticgroups, non-phenolic linkers (derivatives of N-succimidyl-benzoates;dodecaborate), chelating moieties of both macrocyclics and acyclicchelators, such as derivatives of1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA),derivatives of diethylenetriaminepentaacetic avid (DTPA), derivatives ofS-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triaceticacid (NOTA) and derivatives of1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA) and otherchelating moieties. The use of such linkers may be particularlyappropriate in circumstances wherein the agent comprises or consists ofan antibody or fragment thereof as the binding moiety linked, via alinker, to a radioisotope as the therapeutic moiety.

One preferred linker is DTPA, for example as used in ¹⁷⁷Lu-DTPA-11B6.

Optionally, the agent used in the first aspect of the present inventionmay (or may not) further comprise a detectable moiety. For example, adetectable moiety may comprise or consist of a radioisotope, such as aradioisotope selected from the group consisting of: Technetium-99m;Indium-111; Gallium-67; Gallium-68; Arsenic-72; Zirconium-89;Iodine-123, Iodine-124, Iodine-125; Thallium-201. Optionally, the agentmay comprise a pair of detectable and cytotoxic radionuclides, such as⁸⁶Y/⁹⁰Y or ¹²⁴I/²¹¹At. Alternatively, the agent may comprise aradioisotope that is capable of simultaneously acting in a multi-modalmanner as a detectable moiety and also as a cytotoxic moiety to provideso-called “Multimodality theragnostics”. The binding moieties may thusbe coupled to nanoparticles that have the capability of multi-imaging(for example, SPECT, PET, MRI, Optical, or Ultrasound) together withtherapeutic capability using cytotoxic drugs, such as radionuclides orchemotherapy agents. Also included with the present invention is thepossibility of treatment by hyperthermia using high frequencyalternating magnetic fields and accompanied ultrasound imaging. Forexample, see FIG. 11.

Alternatively, the detectable moiety may comprise or consist of aparamagnetic isotope, such as a paramagnetic isotope is selected fromthe group consisting of: gadolinium-157; manganese-55, dysprosium-162,chromium-52; iron-56.

In the case that the agent used in the first aspect of the presentinvention comprises a detectable moiety, then the detectable moiety maybe detectable by an imaging technique such as SPECT, PET, MRI, Opticalor ultrasound imaging.

Therapeutic and detectable moieties may be conjugated or otherwisecombined with the binding moiety using methods well known in the art(for example, the existing immunoconjugate therapy, gemtuzumabozogamicin [tradename: Mylotarg®], comprises a monoclonal antibodylinked to the cytotoxin calicheamicin).

In a further embodiment, the agent used according to the first aspect ofthe invention is used to treat prostate cancer in the form of aformulation comprising a population of agent molecules. In one option,all (or substantially all, such as greater than 90%, 95%, 99%, 99.9% ormore, by weight) of the agent molecules in the population used for thetreatment comprise the same therapeutic moiety. In another option, thepopulation comprises a mixture of other agents with differenttherapeutic moieties. This option will give possibilities to enhance theeffects of targeted radionuclide therapy using various agents suchchemotherapy agents, hormonal therapy agents or other combination oftherapies in which the targeting agent not only delivers therapeuticallyactive radionuclides to tumor associated antigens but alsosimultaneously radiosensitizes the targeted tumor cells by triggering anintracellular signaling cascade. This option is also useful in treatingthe prostate cancer with a mixture of cytotoxic agents, for example,using a cocktail of alpha- and different ranges of beta-emitters, or acocktail of radionuclides with different range, LET (linear energytransfer) and RBE (relative biological effect), for combined treatmentof large tumors, micrometastases, and single tumor cells. In oneembodiment, long-range emitters may be used for treatment of largetumors, and short-range emitters may be used for the treatment ofsmaller tumours such as micrometastases, and single tumor cells.

Optionally, the agent used in the first aspect of the present inventionmay (or may not) further comprises a moiety for increasing the in vivohalf-life of the agent. Exemplary moieties for increasing the in vivohalf-life of the agent may include polyethylene glycol (PEG), humanserum albumin, glycosylation groups, fatty acids and dextran. PEG may beparticularly contemplated.

In an embodiment of the invention, agents comprising a binding agent(e.g., antibody or fragment thereof) that are specific for a kallikreinprotein, such as PSA or hK2, and a therapeutic agent are theninjected/infused into the body. Then the agent binds to tissues thatproduce corresponding antigens, such as PSA or hK2. The biologicstructures, to which the agent becomes bound, may be subsequentlytreated with a suitable agent and/or dosimetry and/or therapy evaluationimaging methods including PET/SPECT/CT/MR/Optical/Ultrasound may beused.

In some circumstances, variations in extent of attenuation of theprostatic cancer cells by the agent may directly correspond toproduction and concentration relations of the target kallikrein (such asPSA and hK2) in the prostatic cancer cells of the patient. Thesevariations may be determined, for example, by the methods of WO2006/08734, the methods of which are incorporated herein by reference,and used to obtain therapeutic information.

For example, pretherapy visualisations of PSA and hK2 antibody bindings,obtained from the imaging methods mentioned above, may be combined withthe methods and uses of the present invention. From the measurement ofattenuations it is possible to directly determine whether theinvestigated tissue is PSA producing, hK2 producing, or both. In lightof this determination it will be possible to tailor the therapy to bemost efficient. Thus individualized patient therapy can be achieved withpre-therapy dose planning. Guidance for individualized patient therapycan be taken from art-known therapies, such as those discussed in (1)Garkavij, et al. (2010) Cancer, 116:1084-1092; (2) Linden, et al. (1999)Clin. Cancer Res., 5:3287s-3291s; (3) Ljungberg, et al. (2003) CancerBiother. Radiopharm., 18:99-107; (4) Minarik, et al, (2010) J. Nucl.Med., 51:1974-1978; (5) Sjogreen-Gleisner, et al. (2011) Q. J. Nucl.Med. Mol. Imaging, 55:126-154; and (6) Sjogreen, et al. (2005) CancerBiother. Radiopharm., 20:92-97; the contents of each of which areincorporated herein by reference.

Accordingly, in one embodiment, the first aspect of the inventioninvolves treating a patient determined to possess PSA-producingprostatic cancer cells with an agent according to the first aspect ofthe present invention that has specificity for PSA.

In another embodiment, the first aspect of the invention involvestreating a patient determined to possess hK2-producing prostatic cancercells with an agent according to the first aspect of the presentinvention that has specificity for human glandular kallikrein (hK2).

In another embodiment, the first aspect of the invention involvestreating a patient determined to possess prostatic cancer cells that areboth PSA-producing and hK2-producing, with an agent according to thefirst aspect of the present invention that has specificity for both PSAand human glandular kallikrein (hK2), or a combination of agents one ofwhich possesses specificity for PSA and the other possessing specificityfor hK2. In the case of combination therapy, the agents may beadministered to the patient separately, sequentially, simultaneously, orformulated as a mixture in the same pharmaceutical composition.

The administration of an agent according to the first aspect of thepresent invention to a patent with prostate cancer may thus result inthe binding of a kallikrein protein, such prostate-specific antigen(PSA; hk3, human kallikrein gene 3) and/or human glandular kallikrein(hK2), present on or in the prostatic cancer cells, and result in theinhibition of growth and/or death of prostatic cancer cells in thepatient. For example, the agent may reduce the rate growth, and/orpresence, of prostatic cancer cells in the patient by at least 10%, inparticular at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and mostparticularly by 100% compared to the observed rate of growth, and/orpresence, of prostatic cancer cells in the patient prior to thetreatment. Methods of measuring the rate of growth, and/or presence, ofprostatic cancer cells in a subject are known in the art.

Thus the invention provides methods for the treatment of prostatecancer.

By ‘treatment’ we include both therapeutic and prophylactic treatment ofthe patient. The term ‘prophylactic’ is used to encompass the use of anagent, or formulation thereof, as described herein which either preventsor reduces the likelihood of prostate cancer, or the spread,dissemination, or metastasis of localised prostate cancer in a patientor subject. The term ‘prophylactic’ also encompasses the use of anagent, or formulation thereof, as described herein to prevent recurrenceof prostate cancer in a patient who has previously been treated forprostate cancer.

The prostate cancer to be treated by the first aspect of the presentinvention may be localised to the prostate, or may be a non-localised(that is, disseminated) prostate cancer. Prostate cancer localised tothe prostate may, for example, be classified as clinical T1 or T2cancers according to the TNM system (abbreviated fromTumor/Nodes/Metastases) whereas non-localised/disseminated prostatecancer may, for example, be classified as clinical T3 or T4 cancers.

The prostate cancer to be treated by the first aspect of the presentinvention may be metastatic prostate cancer. Metastasis refers to thespread of a cancer from its original location to other sites in thebody. For example, the metastatic prostate cancer to be treated may be ametastases present in the lymphatic system; in bone (including spine,vertebrae, pelvis, ribs); metastasis within pelvis, rectum, bladder, orurethra. Metastases present at other less common locations can also betreated with the present invention. The metastases may bemicrometastases. Micrometastase is a form of metastases in which thenewly formed tumors are generally too small to be detected, or detectedwith difficulty. For example, the present invention provides the skilledperson with means to treat single cancer cells or cell clusters, even ifthe presence of such cells or clusters are not possible to diagnose butexist, for example as occult disseminated disease.

Accordingly, it is anticipated that a particularly important technicaladvantage of the treatment provided by the present invention compared tothe prior art treatments of prostate cancer is the enhanced efficacy intreatment of disseminated and/or metastatic (including micrometastatic)prostate cancer.

Thus, in one embodiment, the invention provides agents and methods forpreventing or treatment metastasis of a primary prostate tumour.

Prostate cancer tends to develop in men over the age of fifty, morecommonly in men over 60, 65 or 70, and although it is one of the mostprevalent types of cancer in men, many never have symptoms, undergo notherapy, and eventually die of other causes. This is because cancer ofthe prostate is, in most cases, slow-growing, symptom-free, and sincemen with the condition are older they often die of causes unrelated tothe prostate cancer, such as heart/circulatory disease, pneumonia, otherunconnected cancers, or old age. About two-thirds of prostate cancercases are slow growing, the other third more aggressive and fastdeveloping.

Accordingly, the development of effective treatments of prostate canceris particularly important for management of more aggressive and fastdeveloping forms of the cancer, particularly in younger patient.Accordingly, in one embodiment, the first aspect of the inventionrelates to the treatment of prostate cancer in a patient this is lessthan 70, 65, 60, 55, 50, 45, 40 or less years old at the time ofdiagnosis of prostate cancer and/or at the time of treatment.

Men who have a first-degree relative (father or brother) with prostatecancer are thought to have twice the risk of developing prostate cancer,and those with two first-degree relatives affected are thought to have afive-fold greater risk compared with men with no family history.Accordingly, the first aspect of the invention may relates to thetreatment of prostate cancer in a patient that is characterised in thatone, two, or more, family members, in particular first-degree familymembers (such as a father or brother), has been previously beendiagnosed with prostate cancer.

The first aspect of the invention also relates to the treatment ofprostate cancer in a patient, wherein the prostate cancer to be treatedis castration-resistant prostate cancer (CRPC). CRPC may becharacterised by typically becoming refractory to hormone treatmentafter one to three years, and resuming growth despite hormone therapy.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of an agent as definedabove in respect of the first aspect of the present invention and apharmaceutically-acceptable diluent, carrier or excipient.

Additional compounds may also be included in the pharmaceuticalcompositions, including, chelating agents such as EDTA, citrate, EGTA orglutathione.

The pharmaceutical compositions may be prepared in a manner known in theart that is sufficiently storage stable and suitable for administrationto humans and animals. For example, the pharmaceutical compositions maybe lyophilised, e.g., through freeze drying, spray drying, spraycooling, or through use of particle formation from supercriticalparticle formation.

By “pharmaceutically acceptable” we mean a non-toxic material that doesnot decrease the effectiveness of the kallikrein protein-bindingactivity of the agent of the invention. Such pharmaceutically acceptablebuffers, carriers or excipients are well-known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed.,Mack Publishing Company (1990) and handbook of PharmaceuticalExcipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), hedisclosures of which are incorporated herein by reference).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the agent in the pharmaceuticalpreparation. The diluent may be one or more of saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term “adjuvant” is intended to mean any compound added to theformulation to increase the biological effect of the agent of theinvention. The adjuvant may be one or more of zinc, copper or silversalts with different anions, for example, but not limited to fluoride,chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate,carbonate, lactate, glycolate, citrate, borate, tartrate, and acetatesof different acyl composition. The adjuvant may also be cationicpolymers such as cationic cellulose ethers, cationic cellulose esters,deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationicsynthetic polymers such as poly(vinyl imidazole), and cationicpolypeptides such as polyhistidine, polylysine, polyarginine, andpeptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, glucose, sucrose,mannitol, and cyclodextrines, which are added to the composition, e.g.,for facilitating lyophilisation. Examples of polymers are starch,cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose,alginates, carageenans, hyaluronic acid and derivatives thereof,polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, or for protecting the lipid from chemical and proteolyticdegradation. Examples of lipids are fatty acids, phospholipids, mono-,di-, and triglycerides, ceramides, sphingolipids and glycolipids, all ofdifferent acyl chain length and saturation, egg lecithin, soy lecithin,hydrogenated egg and soy lecithin, which are added to the compositionfor reasons similar to those for polymers. Examples of minerals aretalc, magnesium oxide, zinc oxide and titanium oxide, which are added tothe composition to obtain benefits such as reduction of liquidaccumulation or advantageous pigment properties.

The agents of the invention may be formulated into any type ofpharmaceutical composition known in the art to be suitable for thedelivery thereof.

In one embodiment, the pharmaceutical compositions of the invention maybe in the form of a liposome, in which the agent is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids, which exist in aggregated forms as micelles,insoluble monolayers and liquid crystals. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Suitable lipids also include the lipids above modified bypoly(ethylene glycol) in the polar headgroup for prolonging bloodstreamcirculation time. Preparation of such liposomal formulations is can befound in for example U.S. Pat. No. 4,235,871, the disclosures of whichare incorporated herein by reference.

The pharmaceutical compositions of the invention may also be in the formof biodegradable microspheres. Aliphatic polyesters, such as poly(lacticacid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA)or poly(carprolactone) (PCL), and polyanhydrides have been widely usedas biodegradable polymers in the production of microspheres.Preparations of such microspheres can be found in U.S. Pat. No.5,851,451 and in EP 0 213 303, the disclosures of which are incorporatedherein by reference.

In a further embodiment, the pharmaceutical compositions of theinvention are provided in the form of polymer gels, where polymers suchas starch, cellulose ethers, cellulose carboxymethylcellulose,hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethylcellulose, alginates, carageenans, hyaluronic acid and derivativesthereof, polyacrylic acid, polyvinyl imidazole, polysulphonate,polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropyleneoxide copolymers, polyvinylalcohol/polyvinylacetate of different degreeof hydrolysis, and polyvinylpyrrolidone are used for thickening of thesolution containing the agent. The polymers may also comprise gelatin orcollagen.

Alternatively, the agents may simply be dissolved in saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil),tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of theinvention may include ions and a defined pH for potentiation of actionof the active agent. Additionally, the compositions may be subjected toconventional pharmaceutical operations such as sterilisation and/or maycontain conventional adjuvants such as preservatives, stabilisers,wetting agents, emulsifiers, buffers, fillers, etc.

The pharmaceutical compositions according to the invention may beadministered via any suitable route known to those skilled in the art.Thus, possible routes of administration include parenteral (intravenous,subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar,buccal, oral, parenteral, and rectal. Also administration from implantsis possible. Infusion may be a desired route because of the potentiallyhigh cytotoxicity of the administered agent.

In one embodiment, the pharmaceutical compositions are administeredparenterally, for example, intravenously, intracerebroventricularly,intraarticularly, intra-arterially, intraperitoneally, intrathecally,intraventricularly, intrasternally, intracranially, intramuscularly orsubcutaneously, or they may be administered by infusion techniques. Theyare conveniently used in the form of a sterile aqueous solution whichmay contain other substances, for example, enough salts or glucose tomake the solution isotonic with blood. The aqueous solutions should besuitably buffered (for example, to a pH of from 3 to 9), if necessary.The preparation of suitable parenteral formulations under sterileconditions is readily accomplished by standard pharmaceutical techniqueswell known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Thus, the pharmaceutical compositions of the invention are particularlysuitable for parenteral, e.g., intravenous, administration.

Alternatively, the pharmaceutical compositions may be administeredintranasally or by inhalation (for example, in the form of an aerosolspray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoro-methane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas). In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activepolypeptide, e.g., using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g., sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

The pharmaceutical compositions will be administered to a patient in apharmaceutically effective dose. A ‘therapeutically effective amount’,or ‘effective amount’, or ‘therapeutically effective’, as used herein,refers to that amount which provides a therapeutic effect for a givencondition and administration regimen. This is a predetermined quantityof active material calculated to produce a desired therapeutic effect inassociation with the required additive and diluent, i.e., a carrier oradministration vehicle. Further, it is intended to mean an amountsufficient to reduce and/or prevent, a clinically significant deficit inthe activity, function and response of the host. Alternatively, atherapeutically effective amount is sufficient to cause an improvementin a clinically significant condition in a host. As is appreciated bythose skilled in the art, the amount of a compound may vary depending onits specific activity. Suitable dosage amounts may contain apredetermined quantity of active composition calculated to produce thedesired therapeutic effect in association with the required diluent. Inthe methods and use for manufacture of compositions of the invention, atherapeutically effective amount of the active component is provided. Atherapeutically effective amount can be determined by the ordinaryskilled medical worker based on patient characteristics, such as age,weight, sex, condition, complications, other diseases, etc., as is wellknown in the art. The administration of the pharmaceutically effectivedose can be carried out both by single administration in the form of anindividual dose unit or else several smaller dose units and also bymultiple administrations of subdivided doses at specific intervals.Alternatively, the does may be provided as a continuous infusion over aprolonged period.

The agent defined above in respect of the first aspect of the presentinvention can be formulated at various concentrations, depending on theefficacy/toxicity of the compound being used. The formulation maycomprises the active agent at a concentration of between 0.1 μM and 1mM, between 1 μM and 500 μM, between 500 μM and 1 mM, between 300 μM and700 μM, between 1 μM and 100 μM, between 100 μM and 200 μM, between 200μM and 300 μM, between 300 μM and 400 μM, between 400 μM and 500 μM andabout 500 μM.

It will be appreciated by persons skilled in the art that thepharmaceutical compositions of the invention may be administered aloneor in combination with other therapeutic agents used in the treatment ofa prostate cancer, or before, after or at the same time as the treatmentof the patient with other therapeutic modalities for the treatment ofprostate cancer, such as surgery (e.g., radical prostatectomy),radiation therapy, brachytherapy, external beam radiation therapy,high-intensity focused ultrasound (HIFU), chemotherapy, oralchemotherapeutic drugs, cryosurgery (freezing the tumour), hormonaltherapy (such as antiandrogen therapy), castration or combinations ofthe foregoing.

The present invention also provides a kit comprising an agent as definedabove in respect of the first aspect of the present invention or apharmaceutical composition as defined above.

The present invention also provides an agent for use in medicinesubstantially as described herein.

The present invention also provides a pharmaceutical compositionsubstantially as described herein.

The present invention also provides for the use of an agentsubstantially as described herein.

The present invention also provides a method of treatment substantiallyas described herein.

The present invention also provides a kit substantially as definedherein.

According to one aspect of the invention, a therapeutic method isprovided, which method treat primary and disseminated prostate cancer,with an agent as defined above.

According to another aspect of the invention, a therapy method isprovided, which method may be used to treat metastasis, such as lymphgland metastasis and/or bone metastases, including micrometastases.

According to yet another aspect of the invention, a therapeutic methodis provided, which method may be used to together with or after externalradiotherapy, cytostatic, and androgen treatments, or other treatmentsnot coupled to tumor targeting therapeutic antibodies/fragments.

According to specific aspects of the invention, therapy-labelledantibodies, that are specific for PSA and/or hK2, are provided, whichlabelled antibodies are used to treat prostate cancer, i.e., PSA and/orhK2 producing tissue.

According to another aspect of the invention, uses of said methods areprovided.

The therapy method according to the present invention has the advantageover the prior art that it allows for therapy of prostate cancer, andsaid therapy method may also be used to treat metastasis, includingmicrometastases, such as lymph gland metastasis, or any of the otherforms of metastasis as described above, and be used together with orafter post operative procedure, and during or after radiation,cytostatic, and androgen treatments.

The foregoing description focuses on embodiments of the presentinvention applicable to a therapeutic method of prostatic cancer.However, it will be appreciated that the invention is not limited tothis application but may be applied to many other therapy combinationsincluding for example metastasis, post operative examinations, andexaminations during or after radiation, cytostatic, and androgentreatments. In respect of therapy of metastasis the metastases will betreated in lymph glands.

In another embodiment RadioGuided Surgery (RGS) or Image-Guided Surgery(IGS) may be used to identify tracer-labeled anti-kallikrein specificbinding moieties as described above (such as PSA and/or hK2-antibodies)during and/or before surgery. Thus, an agent comprising a binding moietyand a detectable moiety as discussed above may be administered duringand/or before surgery. In this embodiment the agent, such as a tracerlabeled anti-PSA and/or anti-hK2-antibody, may be first infused.Thereafter, RGS/IGS may be used to identify PSA/hK2 producing tissuewith a detection instrument sensitive to the detectable moiety, duringor before surgery. The detectable moiety may, for example, be aradiation emitting or magnetic-sensitive detectable moiety; it may, forexample, be an emitter of Cerenkov radiation and/or Bremsstrahlung; itmay be a fluorescent label and/or a magnetic or magentizable label.Accordingly, the RGS/IGS according to the present invention may, forexample, be a method that is based on the detection of optical,Cerenkov, Bremsstrahlung, or beta radiation; the detection of aradionuclide label, and/or may involve magnetometry. RGS is well knownto the person skilled in the art as a surgical technique that enablesthe surgeon to identify tissue “marked” by the detectable moiety.

The visualisations obtained according to above may be combined withother radiological visualisation methods, such as SPECT/PET, computedtomography (CT), ultrasound (US), and magnetic resonance tomography(MRT).

Accordingly, in a second aspect, the present invention also provide anagent comprising or consisting of a binding moiety with specificity fora kallikrein protein (such as described above in respect of the firstaspect of the present invention) and a detectable moiety as discussedabove in respect of the first aspect of the present invention for use inmedicine by administration to a patient with prostate cancer before orduring the surgery, such as RadioGuided or Image-Guided Surgery.

Thus, the second aspect also provides for the use of an agent comprisingor consisting of a binding moiety with specificity for a kallikreinprotein and a detectable moiety in the manufacture of a medicament foradministration to a patient with prostate cancer before or during thesurgery, such as RadioGuided or Image-Guided Surgery.

The second aspect of the present invention also provides a methodsurgery, such as RadioGuided or Image-Guided Surgery, that is performedon a patient with prostate cancer, the method comprising the step ofadministering an effective amount of an agent comprising or consistingof a binding moiety with specificity for a kallikrein protein and adetectable moiety to the patient before or during the surgery.

It is contemplated that any method, agent or composition describedherein can be implemented with respect to any other method, agent orcomposition described herein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with the abovedescription and the accompanying drawings. It should be understood,however, that the above description, while indicating variousembodiments of the invention and numerous specific details thereof, isgiven by way of illustration and not of limitation. Many substitutions,modifications, additions and/or rearrangements may be made within thescope of the invention without departing from the spirit thereof, andthe invention includes all such substitutions, modifications, additionsand/or rearrangements.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the kinetics of ¹²⁵I-labelled PSA30 antibody in varioustissues following intravenous administration in normal mice.

FIG. 2 shows the kinetics of ¹²⁵I-labelled PSA30 antibody in varioustissues following intravenous administration in mice implanted withxenograft of metastatic prostate tumour cells, and this shows thatmetastatic prostate tumour cells show a strong take up the PSA30antibody.

FIG. 3 shows Tumor-to-Organs ratios of ¹²⁵I-PSA30 after intravenousinjection in nude mice bearing LNCaP-based subcutaneous tumors atvarious times after injection (n=34). Higher ratio values indicate agreater specificity of uptake in tumour than in the identified tissue.

FIGS. 4A-4H shows the results of digital autoradiography: Individuallynormalized uptake of ¹²⁵I-PSA30 (FIG. 4A) and ¹⁸F-choline (FIG. 4B), 48h post injection of ¹²⁵I-PSA30 plus 1 h post injection oflabeled-choline, in the same tumor section separated by isotope.Histological analysis via H&E (FIG. 4C, FIGS. 4E-4F) and PSA expressionusing 2E9 total PSA antibody (FIG. 4D, FIGS. 4G-4H) were verified usingadjacent sections. There is no direct association between areas of highPSA30 mAb uptake and high choline uptake. Note: this mouse was allowedfree movement after injection of 18F-choline. 209×297 mm (300×300 DPI)

FIG. 5 shows the kinetics of ¹²⁵I-labelled 5A10 antibody in varioustissues following intravenous administration in normal mice.

FIG. 6 shows the kinetics of ¹²⁵I-labelled 11B6 antibody in varioustissues following intravenous administration in normal mice.

FIG. 7 shows the kinetics of ¹²⁵I-labelled 11B6 antibody in varioustissues following intravenous administration in mice implanted withxenograft of metastatic prostate tumour cells. Organ uptake expressed as% IA/g over time.

FIG. 8 shows the biodistribution of ¹¹¹In-11B6 in LnCAP xenografts.Accumulation of radioactivity peaked after 48 hpi with 16.4±1.92% IA/g(percent injected activity per gram). Uptake in normal organs (liver,spleen, kidneys, bone, prostate, testes) are at a lower level. Somewhatelevated uptake was observed in the salivary glands, likely due to acertain normal expression of hK2 expression.

FIG. 9 shows examples of some therapeutic radionuclides.

FIG. 10 shows an illustration of the principle of PAT. In the presenceof low-dose external radiation, a high Z tumour-targeting agent producesa large local absorbed dose enhancement in targeted tumour cells.

FIG. 11 shows an example of how nanoparticles can be used formultimodality imaging and therapy by attachment to tumor targetingagents as antibodies.

FIG. 12 shows the tumor/blood ratios. The ratio increases over time,indicating an active targeting of hK2 with ¹¹¹In-11B6 in LnCAP tumors.

FIG. 13 shows the comparative biodistribution of ¹¹¹In-11B6 inhK2-expressing xenografts (LnCAP) and hK2-negative xenografts (DU145) at48 hpi. Results showed a statistical significant difference (p<0.005)between the two xenografts in the tumor accumulation, while theradioactivity accumulation in most normal organs remained on the samelevel. LnCAP had more than 3-fold higher tumor uptake than the DU145.This indicates that the ¹¹¹In-11B6 is hK2-specific.

FIG. 14 shows the amino acid sequence and epitope structure of PSA,according to Leinonen, J. et al. Clin Chem 2002; 48:2208-2216.

FIG. 15 shows a Scatchard's plot of an exemplary 5A10-Fab.

FIG. 16 shows the labelling kinetics of ¹⁷⁷Lu-11B6.

FIG. 17 shows the in vitro stability of ¹⁷Lu-11B6 in PBS and EDTA.

FIG. 18 shows representative SPECT images of ¹⁷⁷Lu-11B6 in LnCAPxenografts.

FIG. 19 shows the biodistribution of ¹⁷⁷Lu-11B6 in LnCAP xenografts.

FIG. 20 shows the detailed biodistribution at 72 h pi of ¹¹¹Lu-11 B6 inLnCAP xenografts FIG. 21 shows the in vivo biokinetics of ¹⁷⁷Lu-11B6 inLnCAP xenografts.

FIG. 22 shows representative photographs of tumour size before (leftimage) and after (right image) treatment with ¹⁷⁷Lu-11B6.

FIGS. 23A-23C show a summary of the effect of (FIG. 23A) single dose¹⁷⁷Lu-11B6, (FIG. 23B) double dose ¹⁷⁷Lu-11B6 and (FIG. 23C) controltreatment on tumour size in LnCAP xenografts.

FIGS. 24A-24B shows (FIG. 24A) tumour growth data and (FIG. 24B) a SPECTimage for one LnCAP xenografts mouse treated with a single dose¹⁷⁷Lu-11B6

The following examples are included to demonstrate particularembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutespecific modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1—BIODISTRIBUTION OF ¹²⁵I-PSA30 and ¹²⁵I-11B6

Material and Method: The PSA30 and 11B6 antibody were labeled with ¹²⁵I(PerkinElmer, USA), using the Iodogen method. Briefly, a coated testtube with 150 μg 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril was usedfor labeling of 200 μg PSA30. After the mixture had been incubated for15 min at room temperature, low molecular weight components were removedby gel filtration (PD-10 column, GE Healthcare, UK). The radiochemicalpurity was 95% after gel filtration.

Results and discussion: See FIGS. 2 and 3. LNCaP tumors had higheruptake compared to other investigated organs at most time-points andpeaked (4.32% IA/g) at 24 h after intravenous injection of ¹²⁶I-PSA30formulations. By contrast to all other organs showing a decrease ofactivity, LNCaP tumors showed a marked increase of activity (by 32%)during the first 24 hours after injection In comparison to non-tumorbearing mice, thyroid accumulation was greatly augmented. ¹²⁶I-PSA30 mAbuptake in LNCaP tumors peaks at 24 h post injection, with a subsequentsharp decrease in tumor uptake noted by 72 h post injection.Importantly, at this same time point, there is a sharp increase inthyroid uptake. This inverse correlation is a likely indicator that adehalogenation effect has occurred. In conclusion, ¹²⁶I-PSA30 caneffectively target fPSA in LNCaP-based xenograft mice.

EXAMPLE 2—BIODISTRIBUTION OF ¹¹¹In-DTPA-11B6

Material and Method: The animal experiments were performed in accordancewith national legislation on laboratory animals' protection. The animalstudy has been approved by the local Ethics Committee for AnimalResearch. Male immunodeficient nude mice (6-8 wk old) purchased fromTaconic Europe (Bomholt, Denmark) were used for this study. Xenograftsof hK2-expressing LnCAP prostate carcinoma cells were subcutaneouslyimplanted in the right flank. For xenografting, LNCaP cells, 5.2×10⁶cells/mouse in 100 μL cell medium and 100 μL Matrigel (BD Sciences,Bedford, USA). DU145 cells (a hK2-negative cell line), 1-2×10⁶, were scimplanted in the right flank to serve as a negative control. Three-sixweeks post injection of LNCaP cells, 5 groups of mice (4-5animals/group) carrying LNCaP xenografts and 1 group (3 animals/group)carrying DU145 xenografts were each iv. injected with 100 μl¹¹¹In-DTPA-11B6 (-200 kBq in 100 μl and 22.5 μg protein). Animals weresacked at a different time point, 4 h, 24 h, 48 h, 72 h or 168 h p.i.and the control group at 48 h p.i. Organs of interest (see table) wereplaced in 20 ml vials for scintillation counting (Zinsser Analytic,Frankfurt, Germany) weighed and measured in an Automated gamma-counterwith 3-inch NaI(TI) detector (1480 Wizard OY, Wallac, Turku, Finland).All organs were measured twice after dissection and after the last timepoint. Measurement of radioactivity was performed as a standard protocolbelow.

Measurement of Radioactivity: A standard protocol for measurement of aradionuclide was used. Counts per minute corrected with background levelwere used for the evaluation. The tissue uptake value, expressed aspercent injected dose per gram tissue (% ID/g), was calculated as:

% ID/g=(tissue radioactivity/injected radioactivity)/organ weight×100wherein for iv injections:

Injected radioactivity=Average radioactivity in controlsyringes−radioactivity in used syringe−radioactivity in tail

Results and discussion: See FIGS. 8, 12 and 13. Preliminary resultsshowed that ¹¹¹In-11B6 was effectively accumulation in the tumor overtime, peaking at 48 hpi with 16.4±1.92% IA/g (percent injected activityper gram). Radioactivity uptake in normal organs (liver, spleen,kidneys, bone, prostate, testes) are at a lower level. Somewhat elevateduptake was observed in the salivary glands, likely due to a certainnormal expression of hK2 expression (FIG. 8). This will be furtherinvestigated in future studies. Further, the ¹¹¹In-DTPA-11B6 washK2-specific since it was significantly lower uptake in the negativecontrol xenografts DU145 (FIG. 13). The tumor/blood ratio was increasingover time, indicating that more and more of ¹¹¹In-DTPA-11B6 taken up inthe tumor (FIG. 12). In conclusion, the biodistribution data of¹¹¹In-11B6 shows promising tumor-targeting properties in prostatecancer, indicating potential for therapy of prostate carcinoma usingother radionuclides.

EXAMPLE 3—DIGITAL AUTORADIOGRAPHY IMAGING

Materials and methods: DAR was performed on animals injected with¹²⁵I-PSA and ¹⁸F-choline. Animals were euthanized one hour postinjection of metabolic probes and tumors were immediately removed,secured in Cryomount (HistoLab products AB, Sweden), quickly frozen inliquid nitrogen and cut into 100 μm sections for DAR or 20 μm sectionsfor histopathology and immunohistochemistry (IHC) analysis. A siliconstrip detector based system, (Biomolex 700 Imager; Bimolex AS, Oslo) wasused to image the distribution of radioactivity within the thickersections. Differences in both emission spectra and rate of decay wereused to produce separate images of each radionuclide in animals injectedwith more than one radionuclide, in this case, ¹²⁵I and ¹⁸F.

Results and discussion: See results in FIGS. 4A-H. Based on these data,we demonstrate that the PSA30 mAb uptake in excised tumors peaked at 24hours post intravenous injection, and is retained in tumor as comparedto normal tissues. The relatively low T/O ratios (see Table in FIG. 3)can be attributed to factors; such as: a binding site barrier, seen whena low antibody dose is saturated by the fPSA antigens in theperivascular space thus preventing deeper penetration into the solidtumor; insufficient vascular permeability inside of the tumor; ordeiodination of the antibody (as suggested by the high iodineaccumulation in the thyroid). Two ways to improve the T/O ratios wouldbe to increase the antibody dose and test different radiolabels. Despitethis drawback, we found an accumulation of ¹²⁵I-PSA30 activity in tumortissue.

Immunohistochemistry and histopathology (see results in FIGS. 4A-H). Tostudy PSA, 20 μm tumor cryosections (frozen and secured as describedabove) were examined using IHC. The immunoreactivity against PSA or hK2was visualized by use of the DAKO EnVision Flex/HRP system kit (DakoCorporation). Adjacent tumor sections were also stained with hematoxylin(nuclei stain) and eosin (cytoplasmic stain) (H&E) and the generalmorphology analyzed under a standard transillumination microscope. WithH&E staining, viable regions of the tumor sections and necrotic areaswere stained. As a positive control, LNCaP tumor sections were incubatedwith PSA mAb 2E9 at a dilution of 1:1000 and visualized as describedabove. As a negative control, tumor section from a mouse that receivedan intravenous injection of PSA30 was visualized without incubation of asecondary antibody, but including all other steps of IHC. The stainedsections were scanned using a Carl Zeiss MIRAX Scan microscope scannerand viewed with the MIRAX Viewer software (Carl Zeiss Imaging SolutionsGmbH, Germany).

EXAMPLE 4—RADIOLABELING

Direct iodination (²⁵¹I/¹²⁴I/¹³¹I/): Proteins (10 μl, 1 mg/ml in PBS)were mixed with ¹²⁵I as NaI solution (4 MBq) using the Chloramine-T(CAT, Sigma St. Louis, Mo., USA) method. The reaction was initiated byadding CAT in PBS (10 μl, 2 mg/ml) and incubated for 1 min duringvigorous shaking and then terminated by adding sodium metabisulfite (20μl, 2 mg/ml). Labeled proteins were separated from non-reacted ¹²⁵I andlow-molecular-weight reaction components by size-exclusionchromatography on a NAP-5 column (Sephadex G-25, GE Healthcare)pre-equilibrated with PBS.

Indirect iodination (¹²⁵I/¹²⁴I/¹³¹I/²¹¹At): Labeling precursor,N-succinimidyl p-(trimethylstannyl)benzoate (SPMB), was preparedaccording to Orlova et al in Nucl Med Biol 27:827-835 (2000), and 5 μgof SPMB was added to 5 MBq of ¹²⁵I or ²¹¹At in a 5% solution of aceticacid. To start the reaction, 40 μg of chloramine-T (Sigma, St. Louis,Mo.) in aqueous solution was added. The reaction mixture was agitatedfor 5 min, and 80 μg of sodium-meta-bisulphate (Aldrich, Steinheim,Germany) in aqueous solution was added to stop the reaction. Theradiolabeled precursor was added to 40 μg of protein solution in 0.07 Mborate buffer, pH 9.2. The coupling reaction was performed at roomtemperature for 45 min with continuous shaking. Labeled protein variantswere separated from low molecular weight products using a NAP™-5 sizeexclusion column (GE, Healthcare) equilibrated with PBS. Theradiolabeled protein variants were then analyzed an IRMA test (accordingto Evans et al, submitted to CBR) to verify that the labeling procedurehad not affected the binding affinity towards its target.

Radiolabeling with ¹⁷⁷Lu: Conjugation ofisothiocyanate-benzyl-CHX-A″-DTPA (130 nM) to protein (60 nM) wasperformed in 220 uL 0.7 M borate buffer pH 9.2 overnight in a 37° C.water bath. The conjugated CHX-protein was purified on a NAP-5 sizeexclusion column (GE Healthcare, Uppsala, Sweden), using 0.2 M sodiumacetate buffer pH 5.5 as eluent, and then split into ten batches whichwere later used for chelation. Chelation time was optimised by samplingan ongoing chelation process and checking the purity of the chelate oninstant thin-layer chromatography (ITLC) SG plates (Biodex) with 0.2 Mcitrate running buffer. The plates were analysed on a CyclonePhosphorimager (Perkin Elmer, Wellesley, Mass., USA). Chelation wasfound to be complete after 30 min at room temperature. The amount ofradioactive lutetium was varied depending on the needs of individualexperiments.

To test for presence of weakly chelated ¹⁷⁷Lu, EDTA challenges wereperformed. Triplicate samples of the chelated product were challengedwith 200:1 or 1,000:1 molar excess of EDTA versus chelator in a 37° C.overnight incubation. The EDTA concentration was calculated on theassumption that the conjugation was quantitative, thus yielding a meanvalue of two CHX-A-DTPA molecules per antibody. Samples of the solutionswere then analysed by ITLC as above. As a control, triplicate samples of[¹¹¹Lu]-protein were also kept in PBS at 37° C. or 4° C. overnight.

EXAMPLE 5—IN VIVO STABILITY

To analyse the in vivo stability of the radiolabeled conjugates, normalmice are i.v. injected with radiolabels and euthanized after differenttimepoints. Blood is then collected, centrifuged at 5,000 g. Samples ofblood are then separated on NAP-5 columns (cutoff, 5 kDa) equilibratedwith PBS, and the relative amount of radioactivity present thehigh-molecular-fraction is determined.

EXAMPLE 6—CELL SURVIVAL FOR MONITORING THERAPY EFFECTS

Cells are seeded in Petri dishes (diameter 6 cm, approximately 2×10⁵cells/dish). After 48 hours, radiolabelled proteins (57 ng/dish, or 287ng/dish, corresponding to approximately 1:1 and 5:1 antibodies perantigen) are added to the cells. In order to determine the effect of^(125/131)I/¹⁷⁷LU in the media, some of the dishes are preincubated withan excess amount of unlabeled protein (29 μg/dish). Extra dishes areused for estimation of number of decays per cell (DPC). In these dishes,the cellular uptake of radiolabelled protein is measured at six timepoints during the 24-hours incubation. The cells are then washed sixtimes with cold serum-free medium, and the incubation is continued infresh culture medium. Cells are counted approximately once a week, andare reseeded every 2 weeks. The DPC are estimated by calculating thearea under the uptake curve for the two antibody concentrations, as wellas for the blocked dishes. For the lowest radiolabelled proteinconcentration, the cells receive approximately 56 DPC, and for thehighest approximately 150 DPC, whereas the blocked cells receive about 2DPC. The results obtained are analyzed by nonlinear regression(exponential growth), using 1/Y2 as the weighting factor

EXAMPLE 7—IN VIVO STUDIES

The following xenograft models are used: LnCAP, DU145, PC-3 tumormodels. PSA is expressed by all three cell lines while hK2 is expressedby LnCAP and not expressed in DU145 or PC-3.

For xenografting, LNCaP, DU145 or PC-3 cells (2-10 million cells),harvested in 0.02% trypsin/PBS were resuspended in media and injected scinto the right flank with 200 μL of cell suspension containing an equalamount of Matrigen (BD Biosciences, Bedford, Mass.). Tumor formation wasmonitored visually or by palpation.

Blocking Experiment: The blocking experiment in BiodistributionExperiment I was performed in order to establish whether uptake ofradiolabelled proteins in tumors was hK2-specific or not. Before themajor iv injection of radiolabelled protein, 0.8-3.0 mg of unlabeledprotein was iv injected in the blocked mouse group. Uptake ofradioactivity at 24-72 h post injection between the unblocked andblocked groups were compared.

Optimization of specific activity: This experiment is conducted todetermine the influence of specific activity (i.e., the injected proteindose of the radiolabelled conjugate) on the tumor uptake. A series of¹⁷⁷Lu-labelled protein with various predetermined specific activitiesare prepared. An aliquot of ¹⁷⁷Lu-labelled protein is diluted with astock solution of unlabeled protein to provide injection doses varyingfrom 10 μg to 500 μg per LnCAP-bearing mouse. Two-three days afterinjection, the animals are euthanized. Organs and tumors are excised andmeasured for radioactivity uptake. The specific activity providing themost optimal tumor uptake is further considered for dosimetry

Example of dosimetry determination: LnCAP-bearing mice (4 mice/groups)are injected with ¹⁷⁷Lu-labelled protein. The animals are euthanized 4hpi-2 weeks post-injections. Absorbed dose to different organs iscalculated using MIRD scheme. Time-activity curves will be obtained forall organs tissues of interest in the body (animal). The studies will bebased on quantitative imaging from SPECT and/or PET. Integration of thecurves will give cumulated activity. Using the MIRD formalism of basedon own calculated (based on specific geometries and Monte Carlotechniques for absorbed fractions) S values will be used to convertcumulated activity to absorbed dose. In many cases the cross dose has tobe carefully calculated meaning that Monte Carlo based dosimetrycalculations will be done (Hindorf, et al. (2004) J. Nuc. Med.,45:1960-1965; Larsson, et al. (2007) Cancer Biotherapy &Radiopharmaceuticals, 22:438-442; Larsson, et al. (2011) Acta Oncol.,50:973-980).

Example of SPECT and PET imaging: PET-CT and SPECT-CT imaging is anintegral part of radionuclide therapy. It gives an idea of the extent towhich the radioactive material accumulates in the tissues and helps toprovide an estimate of the required therapeutic dose and its effects.For good treatment results, a sufficient dose of radiation must bedelivered to the tumor. This is confirmed by imaging, as discussed inthe references mentioned above in respect of dose planning, the contentsof which are incorporated herein by reference.

Radiobiology: The specific dosimetry methods based on individualpatient/laboratory animal geometries will be used for a proper dosimetryand can be related to biological effects and give the possibility ofcorrelation with radiobiological effects and for optimized therapy ofindividual patients.

EXAMPLE 8—DETERMINATION OF BINDING AFFINITY SCATCHARD'S METHOD

The binding affinity (Kd) of the produced antibody variants weredetermined to by using a Scatchard's method according to Scatchard, AnnN Y Acad Sci 51:660-72 (1949).

In brief, a fixed concentration of antibody (or, in this case, a Fabantibody fragment) and varying concentrations of Eu³⁺-labelled PSAtracers were used.

Surface Plasmon Resonance

The binding kinetics and affinity of the antibody variants may also bedetermined by real-time biospecific interaction analysis on a Biacoreinstrument. In brief, PSA or hK2 is immobilized on a CM5 sensor chip byamine coupling and the immobilization levels reached 1000-2000 responseunits. The different anti-PSA or anti-hK2 antibody derivatives (both mAband Fab) are diluted in concentrations ranging from 0.1-10 nM in HBS-EPbuffer. The binding kinetics are studied in a 5 m in association phaseand a 30 min dissociation phase with a flow rate of 50 μL/min, followedby regeneration. Kinetic constants are calculated using a 1:1 Langmuirbinding model with correction for mass transfer.

EXAMPLE 9—IMMUNORADIOMETRIC ASSAYS (IRMA)

Monoclonal antibody-based immunoradiometric assays (IRMA) forradiolabelled mAb or Fab's binding quality were conducted in triplicateas a four-step sandwich assay with wash steps between incubations(washing buffer: 10 mM Tris-HCL pH 8.0, 0.15 M NaCl, 0.05% Tween 20).The assay was constructed and optimized according to establishedrecommendations. Breakapart microtiter plates were coated with H117 (0.2μg/well), a monoclonal antibody recognizing free or total PSA and humankallikrein 2 (hK2) with the same affinity, 30 diluted in coating buffer(75 mM sodium carbonate pH 9.6) and incubated overnight at 4° C. Thewells were then incubated with 0.2 μg/well quenching buffer (3% fishgelatin in washing buffer) for two hours at room temperature. Next, thewells were coated with 200 μl plasma (female) containing 3 ng/μL fPSAand incubated for two hours at room temperature. Radiolabeled andunlabeled PSA30 were then mixed together in assay buffer (50 mM Tris-HClpH 7.5, 0.1 M NaCl, 5 mM EDTA, 0.25% BSA and 0.05% Tween 20) atdescending concentrations and added to the wells (total volume: 50μL/well). The percentage of labeled antibody per well was as followed:100, 92, 84, 68, 50, 30 and 0 percent. The plates were incubated for twohours at room temperature, washed and measured in a NaI(TI)-well counter(1282 Compugamma CS; LKB Wallac, Turku, Finland). A difference indetection capacity of <25% in relation to theoretical deviance wasaccepted for further application. The estimations of detection qualitypost labeling showed that radiolabeled antibody maintained 70-90% of theaffinity/binding capacity of the unlabeled 0 antibody.

EXAMPLE 10—RADIOIMMUNOTHERAPY WITH 177Lu-m11B6 IN A PROSTATE CANCERMOUSE MODEL

Prostate cancer is the most commonly diagnosed cancer among men in theWestern world, accounting for 25% of all new cases of cancer and for 14%of deaths from cancer (22700443). Current curative treatment strategies(surgery and irradiation) are only successful when the malignancy islocalized to the prostate gland. The therapeutic strategy in the case ofdisseminated disease is limited to castration, which often onlysuppresses growth for 12-18 months before becoming refractory, despitethe hormone-deprived milieu (Scher H I et al, Cancer of the prostate.In: DeVita V T Jr, Hellman S, Rosenberg S A, eds. 7th ed. Philadelphia,Pa.: Lippincott Williams & Wilkins; 2005). Because of the lack oftherapies that have been proven to have an effect beyond a transientresponse, novel molecularly targeted therapies are urgently needed.Because prostate cancer is radiosensitive, it presents an ideal targetfor radioimmunotherapy. Also, radioimmunotherapy typically delivers highlevels of circulating antibodies to bone marrow and lymph nodes, sitesto which the cancer typically spreads. Additionally, radioimmunotherapyemploys a “cross-fire effect”, which, depending on the emitted particlerange of the chosen radioisotope, may kill surrounding antigen-negativebystander cells without direct binding of the antibody (16029058).

Human kallikrein 2 (hK2) is an androgen driven enzyme that is solelyexpressed, at very high concentrations, in healthy and malignantprostatic tissue. Since hK2 has been shown to cleave the zymogen form ofProstate Specific Antigen (PSA), it is believed that one of itsphysiological functions is to act as a regulator of that enzyme. Takentogether, these biological features make hK2 an optimal target in atheragnostic system (therapy and diagnosis).

The aim of this study was to confirm the utility of 11B6, a mAb thatspecifically targets an epitope inside the catalytic cleft of hK2, as avehicle to deliver highly toxic radionuclides specifically to the sitesof prostate cancer growth. In this proof of concept study, we chose tolabel the mAb with 177Lu, a low energy beta particle that also employsgamma emission, enabling SPECT-imaging to be performed.

Materials & Methods

Materials

¹⁷⁷Lu was purchased from Mallinkrodt Medical BV, Petten, Holland. TheCyclone™ Storage Phosphor System and the OptiQuant™ image analysissoftware (Perkin Elmer, Wellesley, Mass., USA) was used to measure theradioactivity on the ITLC (instant thin layer chromatography) strips(Biodex, US) for determining labeling kinetics and radiochemical purity.All chemicals were obtained from Sigma Alchrich and the buffers werein-house prepared using analytical grade water if not otherwise noted.The mAb 11B6 is an antibody specific for the human kallikrein 2 with anaffinity for this antigen of about 1.2 nM; see SEQ ID NOs: 4 and 5 above(obtained from the University of Turku, Finland). For the in vivostudies, the prostate carcinoma cell lines LNCaP expressing hK2 (ATCC,Manassas, Va., USA) were used. Cells were cultured in RPMI 1640 mediumsupplemented with 10% fetal bovine serum and PEST (penicillin 100 IU/mland 100 μg/ml streptomycin). The cells were maintained at 37° C. in ahumidified incubator with 5% CO₂ and were detached with trypsin-EDTAsolution (0.25% trypsin, 0.02% EDTA in buffer, Thermo Scientific).

Conjugation and Radiolabeling

Conjugation of CHX-A″-DTPA with 11B6: A solution of the mAb 11B6 in PBSwas adjusted to pH 9.2 using 0.07 M sodium borate buffer. The sample wasconcentrated on an Amicon Ultra-2 centrifugal filter (2 ml, 100 K). Theprotein solution was conjugated with the chelator CHX-A″-DTPA(Macrocyclics, USA) in a molar ratio of 3:1 chelator to antibody at 40°C. The reaction was terminated after 4 h and CHX-A″-DTPA-11B6, from nowon called DTPA-11B6, was separated from free chelate by size-exclusionchromatography on a NAP-5 column (GE Healthcare) equilibrated with 20 ml0.2 M ammonium acetate buffer, pH 5.5. Conjugated 11B6 and 5A10 waseluted with 1 ml ammonium acetate buffer.

Radiolabeling of DTPA-11B6: DTPA-11B6 in ammonium acetate buffer pH 5.5was mixed with a predetermined amount of ¹⁷⁷LuCl₃. After incubation atroom temperature for 2 h, the labeling was terminated and purified on aNAP-5 column, equilibrated with PBS. Labeling efficiency and labelingkinetics were monitored with ITLC strips, eluted with 0.2 M citric acid.In this system, the radiolabelled conjugate remains at the origin line,while free Lu-177 migrates with the front of the solvent. Theradioactivity distribution was determined with a PhosphorImager system(Perkin Elmer, Wellesley, Mass., USA) using the Optiquant asquantification software (Perkin Elmer, Wellesley, Mass., USA).

Surface Plasmon Resonance

The protein hk2 (Department of Biotechnology, Turku, Finland) in 10 mMNaAc-buffer, pH 4.0, was immobilized on a CM4 research grade chippurchased from Biacore by amino coupling using N-Hydroxysuccinimide(NHS), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) and 1 M Ethanolamine hydrochloride-NaOH pH 8.5 in a Biacore 2000system. The mAb 11B6, its conjugate DTPA-11B6 and Herceptin as anonspecific reference mAb, all in Biacore buffer was flown over two flowcells in five different concentrations (0.5 nM, 5 nm, 10 nM, 50 nM and100 nM) to detect eventual binding. One of the two flow cells containedimmobilized hK2 and the other was a blank reference. The chip wasregenerated using 25 mM Glycin buffer, pH 2.7.

In Vitro Stability Studies

Stability of the labeled conjugates was tested in PBS and in an excessof EDTA, 500× more EDTA then DTPA conjugated on m11B6, incubated at 4°for 1 week and 2 weeks and were analyzed using ITLC strips, see above.

Animal Studies

All animal experiments were performed in accordance with nationallegislation on laboratory animals' protection. The animal study has beenapproved by the local Ethics Committee for Animal Research. Maleimmunodeficient nude mice, NMRI, (6-8 wk old) purchased from TaconicEurope (Bomholt, Denmark) were used for this study.

Xenografts of hK2-expressing LnCAP prostate carcinoma cells weresubcutaneously implanted in the right flank and/or left flank at about10*10⁶ cells per injection.

Animals that developed LNCaP tumors were divided into groups andinjected with either the therapeutic agent 177Lu-DTP-11B6 or with acontrol, see Table 1 below:

TABLE 1 Animals Group nr Treatment 5 animals/group 1 NaCl (control) 11groups 2 Unspecific Ab labeled with Total = 55 animals 177Lu-lowabsorbed dose 3 Unspecific ab labeled with 177Lu-high absorbed dose 4Only 177Lu-low absorbed dose 5 Only 177Lu-high absorbed dose 6177Lu-DTPA-m11B6: A/4 7 177Lu-DTPA-m11B6: A/2 8 177Lu-DTPA-m11B6: A 9177Lu-DTPA-m11B6: 2*A 10 177Lu-DTPA-m11B6: 3*A 11 Only m11B6 A = 26.7MBq All animals included were continuously measured and weighed withinan interval of 3-4 days.

Initially some animals got a lower activity (8MBq) of ¹⁷⁷Lu-DTPA-11B6for investigation of the localization of the therapeutic agent usingSPECT. One mouse from group 8 was also studied with SPECT. These animalshad their organs removed and an automated NaI(TI) well-counter with a3-inch NaI (TI) detector (1480 WIZARD, Wallac Oy, Turku, Finland) wasused to quantify radioactivity in these tissue samples.

To study the effect on the bone marrow blood samples (10 μL) were takenregularly. Blood samples were collected twice a week for 8 weekspostinjection and WBC counts, RBC counts, and platelet counts wereanalyzed in a Medonic Cell Analyzer-Vet CA530 Vet (Boule Medical,Stockholm, Sweden). At the time of blood sampling, the weight andphysical condition of the animals were monitored. Toxicity was evaluatedby monitoring animals for loss of body weight, decline in generalcondition, and hematologic toxicity.

Tumor volume was measured with a caliper. The length I, with w andthickness t were measured and the volume was calculated.

Pharmacokinetics

For the biokinetic study of 111In-m11B6, mice were injected in a tailvein with radionuclide labeled to 25 μg m11B6 antibody. Animals weresacrificed at regular time intervals.

In brief, the mouse mAb 11B6 was conjugated with CHX-A″-DTPA and labeledwith ¹¹¹In forming ¹¹¹In-CHX-A″-DTPA-11B6 (¹¹¹In-DTPA-11B6).Biodistribution studies were performed in an hK2- and AR-positive(LNCaP) PCa xenograft model. DU-145 xenografts (hK2 and AR negative)were used as a control. Animals, NMRI nude, were euthanized atdesignated time intervals, dissected and had organs removed for activitymeasurement. Micro-SPECT imaging was performed. Tumours were sectioned,stained and autoradiography was performed. Some animals were injectedwith cold mouse mAb 11B6 prior to injection of ¹¹¹In-DTPA-11B6 to blockthe uptake of radiolabelled 11B6.

The biokinetics of 177Lu-m11B6 was obtained in the same way as for the111In-m11B6 study.

Data Acquisition and Dosimetry

To determine the absorbed dose to the different target organs, theMIRD-scheme (1) was applied together with mouse-specific S-factors. Thenumber of disintegrations (cumulated activity) was derived from thekinetic data with ¹¹¹In-m11B6. Bi-exponential functions were fitted tothe data points by a least-square algorithm, and the numbers ofdisintegrations were calculated as the integral of these expressionsmultiplied with the decay-factor. The cumulated activity for the bonemarrow was based on the blood method (2), where the activityconcentration in red marrow is supposed to be proportional to theactivity concentration in blood. This red marrow to blood ratio (RMBLR)has been suggested to be 0.36 (2), which was also used in this study.

To determine the mouse-specific S-factors, a version of the MOBY (3)phantom was used in which the organ sizes could be specified. Theaverage weight of the dissected organs from the kinetic-study wasspecified together with the average total weight. The rendering of theflexible NURBS surfaces then generates a strain-specific phantom. Thephantom is voxelized in 160*160*400 voxels. A subcutaneous tumor wereadded on the left flank, by the representation of a sphere outside thenormal skin-contour, but as an ellipsoid with a short axis half to thesphere radius perpendicular to the skin-contour, and the long axis beingas the sphere radius. The salivary gland and the prostate gland weremanually added to the phantom and represented as spheres with radiuscorrelated to the average weights of the organs.

The phantom then acted as input for Monte Carlo simulations of S-factorsfor ¹⁷⁷Lu and ¹¹¹In with the MCNPX 2.6 code as described in earlier work(4).

Therapy Planning

Based on the relationship between absorbed dose and biological effect onthe bone marrow in rats undergoing Radioimmunotherapy with 90Y and 177Lu(Larsson et al., 2012, Med. Phys. 39(7):4434-43) it could be estimatedthat the LD50 for bone marrow would be in the order of 12 Gy. In theliterature LD50 for acute irradiation of rats and mice are the same,about 9 Gy (for example, see Radiobiology for the radiologist, Hall &Giacca (Eds), 2006, 6^(th) edition).

The therapies were then designed from the assumption of a tolerableabsorbed dose of 12 Gy to bone marrow. Then from the dosimetrycalculations the activity corresponding to this absorbed dose wascalculated. The therapy groups were then designed as giving them A/4,A/2, A, 2×A and 3×A. Corresponding activities were used for thecontrols.

Results

Radiolabelling of ¹⁷⁷Lu-DTPA-m11B6

The labeling kinetics results in FIG. 16 show that the labelingefficiency is very high, reaching 90% after 2 hours incubation. Thisensures a likelihood of excellent therapy efficacy with minor effects ofunconjugated 177Lu.

The in vitro stability results in PBS and EDTA show good stability overtime with almost no change with time within two weeks (see FIG. 17).Also, no difference can be seen between PBS and EDTA incubation,indicating a very good conjugation chemistry ensuring stability in vivowith long retention and circulation times.

Imaging

The SPECT images in FIG. 18 show the distribution of ¹⁷⁷Lu-DTPA-m11B6 inxenografted nude mice, (8MBq injected).

The different images of FIG. 18 are as explained in Table 2.

TABLE 2 Column 1 Column 2 Column 3 Column 4 S1: 48 h S1: 72 h S2: 72 hS3: 72 h S11: 72 h DU 145 S1: 168 h S2: 168 h S8: 168 h Blocked S9: 168h Blocked

The first column for mouse S1 shows the excellent uptake in the tumor inmouse S1 with an increased uptake with time 48, 72 and 168 h pi. Thesecond column shows mouse S2 at 72 and 168 h with same high tumoruptake. Column 3, row 2 shows mouse S3 at 72 h pi with similar hightumor uptake. Row 3, column 3 shows mouse S8 at 168 h pi with no tumoruptake after blocking with cold antibodies showing the specificity ofm11B6 for tumor targeting. Similar results for mouse S9 in column 4, row3. Finally mouse S11 in column 4, row 2 shows no uptake in tumor of cellline DU 145 not specific for the m11B6 antibody.

These results demonstrate the high specificity of m11B6 resulting inhigh tumor accumulation.

Biodistribution

The results of the biokinetic study of the 1111n-m11B6 are discussed inExample 2 above. An accumulation is seen in the tumor tissue with amaximum of 16% IA/g at 48 hours; all other organs show a decline ofactivity except the salivary glands (see FIG. 8). Thus, a high tumor tonormal organ ratio is obtained, which is a prerequisite for high therapyefficacy.

Biodistribution data for 177Lu-m11B6 (at 72 h and 168 h) is shown inFIG. 19. Here, a much higher accumulation of activity can be seencompared to ¹¹¹In-m11B6, with almost 30% IA/g at 168 h. This furtherunderlines the feasibility of high therapeutic efficacy withradiolabelled 11B6 antibody.

The detailed results at 72 h pi of blocking together with using tumorcell line DU-145 is shown in FIG. 20. Here, the distribution of the¹⁷⁷Lu-DTPA-m11B6 from the SPECT study shown above in mice with LnCaP orDU-145 and blocking the hK2 Ag with preinjection of non-conjugated 11B6are given. As seen in detail the blocking and the tumor cell line DU-145result in no uptake in the tumors showing the high specificity of m11B6.

Dosimetry

FIG. 21 shows the results of the biokinetic study of 111In-m11B6 usedfor the dosimetry calculations. In each graph within the compositefigure, the upper dotted line represents the results of the kineticstudy with one standard deviation, the solid curve is an adaptedbi-exponential function and the lower dotted curve is when the decay of111In is considered. The area under the lower dotted curve is thecumulated activity used in the dosimetry calculations.

Based on the biokinetics as shown in FIG. 21, the cumulated activitieswere calculated. Using the 111In S values, the absorbed dose peractivity unit (Gy/MBq) were then calculated. In Table 3 below are giventhe results for 111In.

Based on the assumption that the same biokinetics can be used for177Lu-m11B6, the corresponding cumulated activities were calculated withits physical half time. When using the S-values for 177Lu, the absorbeddose per activity unit was calculated. The assumption of similarbiokinetics is justified by the results of the uptake of 177Lu-m11B6showing similar uptake values as for 111In-m11B6 (see FIGS. 8 and 19).

TABLE 3 Absorbed dose (Gy/MBq) from therapy with ¹¹¹In- and ¹⁷⁷Lu-11B6¹¹¹In ¹⁷⁷Lu Organ Self-dose Total-dose Self-dose Total-dose Remainder0.072 0.081 0.504 0.516 Blood 0.195 0.235 1.207 1.283 Heart 0.076 0.1330.442 0.622 Lung 0.059 0.106 0.396 0.532 Liver 0.102 0.130 0.636 0.666Spleen 0.082 0.115 0.670 0.716 GI-tract 0.041 0.073 0.246 0.298 Kidney0.088 0.122 0.491 0.525 Thyroid 0.001 0.041 0.006 0.094 Bone 0.020 0.0860.131 0.267 Brain 0.003 0.025 0.017 0.039 Prostate 0.036 0.075 0.2360.295 Testes 0.031 0.061 0.246 0.294 Salivary glands 0.223 0.249 1.8851.926 Tumor 0.294 0.312 2.236 2.252 Bone Marrow 0.063 0.092 0.386 0.452

Based on an LD50 value of 12 Gy of bone marrow, a dose of 26.7 MBq canbe injected. This means an absorbed dose for the tumor of 60 Gy.

Recalculating the tumor absorbed dose (assuming that the 111-In-m11B6kinetics is the same as for 177Lu-m11B6) and changing uptake values at72 h pi (16% IA/g till 20% IA/g) and at 168 h pi (15% IA/g to 28% IA/g)results in the absorbed doses as given in Table 4 below. It can then beseen that the absorbed dose to tumor will increase from 60 Gy to 120 Gy

TABLE 4 Organ Self-dose Total absorbed dose Remainder 0.494456 0.507415Blood 1.20655 1.28288 Heart 0.441901 0.621222 Lung 0.396110 0.530831Liver 0.636344 0.665149 Spleen 0.669825 0.715375 GI-tract 0.2458240.297540 Kidney 0.490722 0.524355 Thyroid 0.00649596 0.0923319 Bone0.131186 0.266107 Brain 0.0170275 0.0386855 Prostate 0.236380 0.293902Testes 0.246161 0.293579 Saliva 1.88491 1.92563 Tumor 4.48777 4.50278Bone marrow 0.386496 0.451228

The above dosimetry calculations are based on a proper dosimetry model;the biokinetics reveal that a therapeutic absorbed dose can be deliveredto the tumors within safe limits for bone marrow toxicity.

Animal Tumor Shrinkage

FIG. 22 shows how the tumor in one of the mice (visible on the animal'sflank, under the skin) decreases in volume following treatment.

Radioimmunotherapy Results

FIG. 23 shows the results for the study groups with administeredactivities (a) D, (b) 2×D and (c) a control group (where D=26.7 MBq).

There is a clear trend of decrease of tumor volume in both treatmentgroups. The onset of tumor shrinkage is seen already a few days afterinjection of 177Lu-m11B6. In the control group there is an increase oftumor volume after the injection of NaI solution.

FIG. 24 (a) shows the results for one of the mice in the group injectedwith activity A. Here, the tumor grows steadily from day one until daysix when activity A of 177Lu-m11B6 is administered. Following treatment,a rapid drop in tumor volume is observed.

In the SPECT study (8 d pi) the tumor volume is shown with stillactivity present; see FIG. 24(b).

CONCLUSION

The present study with exemplary antibody 177Lu-m11B6 clearlydemonstrates a therapeutic efficacy against prostate cancer tumours invivo.

Both theoretical calculations based on the special dosimetry model andthe in vivo measured biokinetics show favorable dosimetry giving a hightherapeutic ratio. This is then verified in the animal study with goodtherapy results showing rapid tumor volume shrinkage.

REFERENCES

-   1. Bolch W E, Eckerman K F, Sgouros G, Thomas S R. MIRD pamphlet No.    21: a generalized schema for radiopharmaceutical    dosimetry—standardization of nomenclature. J Nucl Med. 2009;    50:477-484.-   2. Sgouros G. Bone marrow dosimetry for radioimmunotherapy:    theoretical considerations. J Nucl Med. 1993; 34:689-694.-   3. Segars W P, Tsui B M, Frey E C, Johnson G A, Berr S S.    Development of a 4-D digital mouse phantom for molecular imaging    research. Mol Imaging Biol. 2004; 6:149-159.-   4. Larsson E, Strand S E, Ljungberg M, Jonsson B A. Mouse S-factors    based on Monte Carlo simulations in the anatomical realistic Moby    phantom for internal dosimetry. Cancer Biother Radiopharm. 2007;    22:438-442.-   5. Erik Larsson, Michael Ljungberg, Linda Martensson, Rune Nilsson,    and Jan Tennvall, Sven-Erik Strand and Bo-Anders Jonsson Use of    Monte Carlo simulations with a realistic rat phantom for examining    the correlation between hematopoietic system response and red marrow    absorbed dose in Brown Norway rats undergoing radionuclide therapy    with 177Lu- and 90Y-BR96 mAbs Medical-   6. Linda Martensson, Zhongmin Wang, Rune Nilsson, Tomas Ohlsson,    Peter Senter, Hans-Olov Sjögren, Sven-Erik Strand, Jan Tennvall,    Determining Maximal Tolerable Dose of the Monoclonal Antibody BR96    Labeled with 90Y or 177Lu in Rats: Establishment of a Syngeneic    Tumor Model to Evaluate Means to Improve Radio immunotherapy Clin    Cancer Res 2005; 11:7104s-7108s. 2005.

1.-56. (canceled)
 57. A method for the treatment of castration-resistantprostate cancer (CRPC) in a patient, the method comprising administeringa therapeutically effective amount of an agent comprising (a) anantibody or antigen-binding fragment thereof with specificity for humanglandular kallikrein (hK2) and (b) a cytotoxic moiety comprising ²²⁵Ac,wherein the antibody or antigen-binding fragment thereof withspecificity for hK2 comprises the six complementarity determiningregions (CDRs) of antibody 11B6, wherein the heavy chain of the 11B6antibody comprises SEQ ID NO: 4 and the light chain of the 11B6 antibodycomprises SEQ ID NO:
 5. 58. The method of claim 57, wherein the antibodyor antigen-binding fragment thereof with specificity for hK2 comprisesat least a portion of an antibody constant region derived from a humanantibody.
 59. The method of claim 57, wherein the antibody orantigen-binding fragment thereof with specificity for hK2 is selectedfrom the group consisting of 11B6 and antigen-binding fragments thereof,wherein the heavy chain of the 11B6 antibody comprises SEQ ID NO: 4 andthe light chain of the 11B6 antibody comprises SEQ ID NO.
 5. 60. Themethod of claim 57, wherein the antibody or antigen-binding fragmentthereof with specificity for hK2 is linked indirectly to the cytotoxicmoiety.
 61. The method of claim 57, wherein the antibody orantigen-binding fragment thereof with specificity for hK2 is linkeddirectly to the cytotoxic moiety.
 62. The method of claim 57, whereinthe agent displays tumour uptake characteristics substantiallyequivalent to the tumour uptake characteristics of the antibody orantigen-binding fragment thereof with specificity for hK2 alone.
 63. Themethod of claim 57, wherein the prostate cancer is metastatic CRPC. 64.The method of claim 57, wherein the patient has prostate cancer and isless than 70 years old at the time of diagnosis of prostate cancerand/or at the time of treatment.
 65. The method of claim 57, wherein thepatient is characterized in that a family member has been previouslydiagnosed with prostate cancer.
 66. The method of claim 57, wherein thepatient has previously been treated with one or more prostate cancertherapies.
 67. The method of claim 57, wherein variable frameworkresidues of the antibody or an antigen-binding fragment thereof comprisehuman framework residues.
 68. The method of claim 57, wherein thecytotoxic moiety is linked to the antibody or antigen-binding fragmentthereof via a non-phenolic linker.
 69. The method of claim 57, whereinthe cytotoxic moiety is linked to the antibody or antigen-bindingfragment thereof via a chelating moiety.
 70. The method of claim 69,wherein the chelating moiety is selected from the group consisting ofderivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10, tetraacetic acid(DOTA), derivatives of diethylenetriaminepentaacetic acid (DTPA),derivatives ofS-2-(4-lsothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triaceticacid (NOTA), and derivatives of1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA).
 71. Themethod of claim 57, wherein the method reduces the rate of growth ofprostatic cancer cells in the patient by at least 10% compared to theobserved rate of growth of prostatic cancer cells in the patient priorto the treatment.
 72. The method of claim 57, wherein the method reducesthe rate growth of prostatic cancer cells in the patient by at least20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the observed rateof growth of prostatic cancer cells in the patient prior to thetreatment.
 73. The method of claim 57, wherein the agent is administeredat a dose of about 1 mg/kg.